Cells for drug discovery

ABSTRACT

Disclosed herein are compositions and method useful in screening a compound for its interaction and/or effect with a molecular target and/or cellular process.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to provisional patent applicationSer. No. 60/181,117, filed Feb. 8, 2000, from which priority is claimedunder 35 USC §119(e)(1) and which is incorporated herein by reference inits entirety.

TECHNICAL FIELD

[0002] This disclosure resides in the fields of cellular engineering anddrug discovery.

BACKGROUND

[0003] Much of the research activity of pharmaceutical companies inyears past focused on the incremental improvement of existing drugs.These efforts involved repetitive rounds of compound modification andbiological testing and have resulted in a large percentage of theavailable drugs directed to similar targets.

[0004] Approximately a dozen years ago, the emphasis of pharmaceuticalresearch activities began shifting toward the purposeful discovery ofnovel chemical classes and novel molecular targets. This change inemphasis, and timely technological breakthroughs (e.g., molecularbiology, laboratory automation, combinatorial chemistry) gave birth tohigh throughput screening, or HTS, which is now widespread throughoutthe biopharmaceutical industry.

[0005] High throughput screening involves several steps: creating anassay that is predictive of a particular physiological response;automating the assay so that it can be reproducibly performed a largenumber of times; and, sequentially testing samples from a chemicallibrary to identify chemical structures able to “hit” the assay,suggesting that such structures might be capable of provoking theintended physiological response. Hits from the high throughput screenare followed up in a variety of secondary assays to eliminateartifactual results, particularly toxic compounds.

[0006] A high throughput screen could involve the testing of 200,000compound samples or more, therefore requiring the use of lab robots.Examples of samples tested in such an assay include pure compounds savedin compound archives (e.g., certain pharmaceutical companies havechemical libraries that have been generated through decades of medicinalchemistry effort), samples purchased from academic sources, naturalproduct extracts and libraries purposefully created for high throughputscreening such as combinatorial libraries.

[0007] The assays used in high throughput screens are intended to detectthe presence of chemical samples possessing specific biological orbiochemical properties. These properties are chosen to identifycompounds with the potential to elicit a specific biological responsewhen applied in vivo. High throughput screens typically identify drugcandidates rather than the agents that will ultimately be used as drugs.A compound of a certain chemical class found to have some level ofdesired biological property in a high throughput assay can then be thebasis for synthesis of derivative compounds by medicinal chemists.

[0008] The assays fall into two broad categories: biochemical assays andcell-based assays. Biochemical assays utilize pure or semi-purecomponents outside of a cellular environment. Enzyme assays and receptorbinding assays are typical examples of biochemical assays. Cell-basedassays utilize intact cells in culture. Examples of such assays includeluciferase reporter gene assays and calcium flux assays.

[0009] Biochemical assays are usually easier to perform and aregenerally less prone to artifacts than conventional cell-based assays.Compounds identified as “active” in a biochemical assay typicallyfunction according to a desired mechanism, decreasing the amount offollow-up experimentation required to confirm a compound's status as a“hit.” A major disadvantage of biochemical assays, however, is the lackof biological context. Compound “hits” from biochemical screens do nothave to traverse a plasma membrane or other structures to reach andaffect the target protein. Consequently, biochemical assays tend to befar less predictive of a compound's activity in an animal thancell-based assays.

[0010] Cell-based assays preserve much of the biological context of amolecular target. Compounds that cannot pass through the plasma membraneor that are toxic to the cell are not pursued. This context, however,adds complexity to the assay. Therefore conventional cell-based assaysare much more prone to artifact or false positive results than arebiochemical assays. Compounds that trigger complex toxic reactions ortrigger apoptosis are particularly troublesome. Much of the labordevoted to conventional cell-based high throughput screening is directedto follow-up assays that detect false hits or hits that work byundesirable mechanisms.

[0011] If false positive or artifactual hits could be rapidly identifiedand eliminated, the ease and efficiency of biochemical assays could beapproached in cell-based assays, while preserving the biologicalcontext. The result would be an assay with optimum throughput andoptimum predictability of biological function. In short, a moreefficient process for the discovery of new pharmaceuticals would beproduced.

SUMMARY

[0012] In one aspect, methods of screening a compound for interactionwith a molecular target are provided. In certain embodiments, the methodinvolves the following steps: (a) contacting a first cell with thecompound; (b) determining a first value of a property of the first cell,the property being responsive to the cell being contacted with thecompound; (c) contacting a second cell with the compound, wherein thesecond cell comprises an exogenous zinc finger protein that directly orindirectly modulates expression of the molecular target; (d) determininga second value of the property in the second cell. A difference betweenthe value of the cell property in the first cell and the cell propertyin the second cell provides an indication of an interaction between thecompound and the molecular target. The zinc finger protein preferablymodulates expression of the molecular target itself, but, in someembodiments, may indirectly modulate expression of the molecular targetfor example, by modulating expression of a protein that then modulatesand/or affects the molecular target. Therefore, using these screeningmethods, one can, for example, test a compound for its capacity totransduce a signal through the molecular target or its capacity to blocktransduction of a signal through the molecular target.

[0013] In certain embodiments, the first and second cells aresubstantially identical with the exception that the second cell containsan exogenous zinc finger protein and/or sequences encoding an exogenouszinc finger protein. In certain embodiments, there may be furthergenetic (and/or phenotypic) differences between the first and secondcells.

[0014] In any of the methods described herein, the zinc finger proteincan be a component of a fusion molecule, for example a fusion of a zincfinger protein and a functional domain. The functional domain may be,for example a repression domain such as KRAB, MBD-2B, v-ErbA, MBD3,unliganded TR, and members of the DNMT family; an activation domain suchas VP16, the p65 subunit of NF-kappa B, ligand-bound TR, and VP64; aninsulator domain; a chromatin remodeling protein or component of achromatin remodeling complex; and/or a methyl binding domain. Accordingto the methods, the zinc finger protein either activates or inhibits theexpression of the target. The zinc finger protein (or fusion) canactivate expression, for example, such that the expression level in thesecond cell is more than 125% or 175% of the expression level in thefirst cell. The zinc finger protein can inhibit expression, for example,such that the expression level in the second cell is less than 95%, 75%,50%, 25% or 5% of the expression level in the first cell.

[0015] In certain embodiments, the molecular target is a protein.However, a molecular target is any molecule whose expression can bemodulated by a zinc finger protein, for example, RNA, carbohydrateand/or lipid.

[0016] In any of the method described herein, the zinc finger protein(or fusion molecule) can be provided as a protein or as a polynucleotideencoding the protein or fusion molecule. Thus, according to the methods,the zinc finger protein is either expressed in, or added to, the secondcell. In certain embodiments in which the zinc finger protein isprovided as a polynucleotide, expression of the zinc finger protein canbe inducible, for example using an inducible transcription controlelement (e.g., promoter) operably linked to the sequence encoding thezinc finger protein. In these embodiments, the first and second cellsmay both contain a polynucleotide encoding a zinc finger protein butexpression is induced in only one of the two matched cells. Furthermore,the first and/or second cells may contain more than one exogenous zincfinger protein or fusion molecule (or polynucleotide encoding same).

[0017] In other aspects, the first and/or second cells used in thescreening methods can also comprise a reporter (e.g., selectablemarker). For example, in certain embodiments, methods are providedwherein a cell comprises a zinc finger protein whose expression isoperably linked to a transcription control element responsive to a knownmolecular target, preferably a component of a cellular process such as abiochemical pathway or signal transduction pathway. Thus, underconditions in which the signal transduction pathway is active, the zincfinger protein is expressed. The cell also preferably comprises apolynucleotide encoding a reporter (e.g., a fluorescent protein, such asgreen fluorescent protein, a luciferase, a beta-galactosidase, abeta-glucuronidase, a beta-lactamase, a peroxidase such as horseradishperoxidase, an alkaline phosphatase, CAT, etc.). A subset of reportermolecules includes selectable markers (e.g., drug resistance, thymidinekinase, etc.). The reporter or selectable marker can be operably linkedto transcriptional control elements that are modulated by the zincfinger protein (or fusion containing the zinc finger protein).Accordingly, when a test compound is administered to the cell, theability of the compound to interact with a target (e.g., a component ofa signal transduction pathway) to modulate production of the ZFP will bereflected in the amount of reporter and/or selectable marker produced.In certain embodiments, the zinc finger protein (or fusion) repressesexpression of the reporter and/or selectable marker. In these cases, ifthe compound interacts with its target in such a way as to block asignal transduction pathway, production of a reporter whose expressionis controlled by the ZFP is increased. The cells may also comprise morethan one reporter and/or selectable marker.

[0018] In additional embodiments, cells which overexpress a moleculartarget are provided, wherein overexpression is mediated by the action ofan exogenous zinc finger protein. Similarly, cells which underexpress amolecular target, wherein underexpression is mediated by the action ofan exogenous zinc finger protein, are provided. Methods of making andusing such cells are also provided.

[0019] In still further embodiments, methods are provided that involveusing one or more cellular components isolated from a cell containing anexogenous zinc finger protein. In preferred embodiments, the cellularcomponent is cell membranes which are preferably isolated from cellsthat overexpress a molecular target (e.g., receptor) by virtue of theactivity of the zinc finger protein (or fusion containing same). Theisolated membranes can be used, for example, for binding studies (e.g.,using radiolabeled ligands).

[0020] In another aspect, cells comprising any of the zinc fingerproteins described herein are provided. In preferred embodiments, a cellthat expresses a molecular target is provided. A cell of this sortexhibits a property that is responsive to the cell being contacted witha compound that interacts with the molecular target. The cell containsan exogenous zinc finger protein that modulates the production of aprotein in the cell, preferably the molecular target.

[0021] In yet another aspect, kits for screening a compound of interestcomprising any of the cells, proteins, polynucleotides and the likedescribed herein are provided. In preferred embodiments, the kit furthercomprises ancillary reagents, instructions and other materials designedto carry out of the methods of screening described herein.

[0022] These and other embodiments will be readily apparent to theskilled artisan in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows the process of assembling a nucleic acid encoding adesigned ZFP.

[0024]FIG. 2, panels A-C depict inducible expression of the endogenousEPO gene in stably transformed 293 cells, in response to synthesis of azinc finger protein (EPOZFP-862) under the control of a tetracyclineresponsive full-length CMV promoter. FIG. 2A shows the doxycycline (Dox)dose-response for EPO expression, as determined by ELISA. FIG. 2B showsan immunoblot analysis of EPOZFP-862 expression in cells treated withthe indicated concentration of doxycycline, using an anti-FLAG antibody.FIG. 2C shows Northern blot analysis of EPO mRNA induced by EPOZFP-862.Lane 1 is a control showing untransfected cells; lane 2 shows cellstransiently transfected with EPOZFP-862; lanes 3 and 4 show cells stablytransfected with EPOZFP-862, in the absence (lane 3) and presence (lane4) of Dox.

DETAILED DESCRIPTION

[0025] Overview

[0026] The compositions and methods disclosed herein include novelassays for screening candidate compounds for their ability to interactwith particular molecular targets. The assays include both cell-basedand biochemical assays. Furthermore, the assays described hereincomprise or are derived from cells comprising a zinc finger protein thatis capable of modulating expression of a gene of interest.

[0027] In one embodiment, zinc finger proteins or polynucleotidesencoding these proteins are introduced into cells (e.g., via stable ortransient transfection) to overexpress a molecular target of interest.The effect of a candidate compound on the molecular target in theZFP-containing cells can be compared to the effect of the same compoundin non-ZFP-containing cells to determine the ability of the compound tointeract with the target. Thus, the ZFP can modulate (e.g., activate)expression of the molecular target itself. Alternatively, the ZFP canmodulate expression of a different molecule which then acts directly orindirectly on the molecular target, for example the ZFP can modulateexpression of an upstream or downstream molecule in a signaltransduction pathway that modulates the molecular target.

[0028] In other embodiments, a gene encoding a ZFP is under the controlof an inducible promoter and both first and second cells comprise theinducible gene. The cells are therefore substantially identical andexpression of the ZFP can be induced in one cell (the test cell) and notin the other cell (the control cell) to determine the interaction of acandidate compound with the molecular target. The cells can be stably ortransiently transfected with the inducible ZFP construct.

[0029] In another embodiment, zinc finger proteins are introduced intocells to overexpress a cell surface or membrane-bound molecule, forexample a receptor. The receptor-rich membranes are then isolated andused for biochemical assays, for example by measuring binding of acompound.

[0030] In yet other embodiments, compounds are tested for their abilityto interact with a molecular target, for example any target involved ina cellular process (e.g., a biochemical pathway or a signal transductioncascade). For example, a test compound is administered to a cell that isknown to express a receptor that initiates a particular signaltransduction cascade. Furthermore, the test cell also comprises (1) apolynucleotide encoding a fusion molecule comprising an exogenous zincfinger protein and a functional domain and (2) a polynucleotide encodinga reporter molecule (e.g., green fluorescent protein, or selectablemarker such as drug resistance). The polynucleotide encoding the fusionmolecule is operably linked to transcriptional control elements that areresponsive to the cellular process of interest. For example, thepolynucleotide encoding the fusion molecule can be operably linked totranscriptional control sequences derived from a gene whose expressionis modulated as a result of the signal transduction cascade. The fusionmolecule can comprise, for example, a zinc finger protein and arepression domain such that, when expressed, it represses expression ofthe reporter molecule. Thus, when the receptor and associated processes(e.g., signal transduction cascade) are functioning, the fusion moleculeis expressed and, in turn, it serves to repress the expression of thereporter molecule. If a candidate test compound interacts with acomponent of the signal transduction pathway so as to block the pathway,expression of the repressive fusion molecule will be diminished oreliminated and the level of reporter will increase. Thus, any compoundcan be screened for its ability to interfere with a signal transductioncascade by monitoring reporter levels.

[0031] The practice of the disclosed methods employs, unless otherwiseindicated, conventional techniques in molecular biology, biochemistry,genetics, computational chemistry, cell culture, recombinant DNA andrelated fields as are within the skill of the art. These techniques arefully explained in the literature. See, for example, Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold SpringHarbor Laboratory Press, 1989; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodicupdates; and the series METHODS IN ENZYMOLOGY, Academic Press, SanDiego.

[0032] The disclosures of all patents, patent applications andpublications mentioned herein are hereby incorporated by reference intheir entireties.

[0033] Definitions

[0034] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide”are used interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form. Forthe purposes of the present disclosure, these terms are not to beconstrued as limiting with respect to the length of a polymer. The termscan encompass known analogues of natural nucleotides, as well asnucleotides that are modified in the base, sugar and/or phosphatemoieties. In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

[0035] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acid.

[0036] A “zinc finger” is a sequence-specific binding domain comprisinga polypeptide sequence (generally approximately 30 amino acids) having astructure that is stabilized by coordination of a zinc atom. A zincfinger can bind DNA, RNA and/or amino acid sequences. With respect toDNA binding, a single zinc finger generally binds to a target subsitecomprising 2 to 4 base pairs, typically 3 or 4 base pairs. An exemplaryclass of zinc fingers has the general structure-Cys-X₂₋₄-Cys-X₁₂-His-X₃₋₅-His- (where X is any amino acid); comprisingtwo cysteine residues and two histidine residues which coordinate thezinc atom (a so-called CCHH or C₂H₂ zinc finger). However, additionalzinc finger structures such as, for example, CCHC, CCCH, CHHC, CHCH andCHHH are also useful in the disclosed methods and compositions.

[0037] A “zinc finger DNA binding protein” is a protein or segmentwithin a larger protein that binds DNA in a sequence-specific mannerthrough the binding activity of one or more zinc fingers. The term zincfinger DNA binding protein is often abbreviated as zinc finger proteinor ZFP. A ZFP can comprise polypeptide domains in addition to zincfingers; for example, other DNA-binding domains and/or functionaldomains.

[0038] A “designed” zinc finger protein is a protein not occurring innature whose design/composition results principally from rationalcriteria. Rational criteria for design include application ofsubstitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP designsand binding data, for example as described in co-owned PCT WO 00/42219and co-owned U.S. patent application Ser. No. 09/444,241 filed Nov. 19,1999.

[0039] A “selected” zinc finger protein is a protein not found in naturewhose production results primarily from an empirical process such asphage display. See e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No.6,007,988; U.S. Pat. No. 6,013,453; WO 95/19431; WO 96/06166 and WO98/54311.

[0040] An “optimized” zinc finger protein refers to a zinc fingerprotein which has been designed or selected as described supra, thentested for binding specificity and altered in sequence to improve itsbinding specificity, as described in co-owned U.S. patent applicationSer. No. 09/716,637, filed Nov. 20, 2000, entitled “IterativeOptimization in the Design of Binding Proteins.”

[0041] The term “naturally-occurring” is used to describe an object thatcan be found in nature, as distinct from being artificially produced byhumans.

[0042] Nucleic acid or amino acid sequences are “operably linked” (or“operatively linked”) when placed into a functional relationship withone another. For instance, a promoter or enhancer is operably linked toa coding sequence if it regulates, or contributes to the modulation of,the transcription of the coding sequence. Operably linked DNA sequencesare typically contiguous, and operably linked amino acid sequences aretypically contiguous and in the same reading frame. However, sinceenhancers generally function when separated from the promoter by up toseveral kilobases or more and intronic sequences may be of variablelengths, some polynucleotide elements may be operably linked but notcontiguous. Similarly, certain amino acid sequences that arenon-contiguous in a primary polypeptide sequence may nonetheless beoperably linked due to, for example folding of a polypeptide chain.

[0043] With respect to fusion polypeptides, the term “operativelylinked” can refer to the fact that each of the components performs thesame function in linkage to the other component as it would if it werenot so linked. For example, with respect to a fusion polypeptide inwhich a ZFP DNA-binding domain is fused to a transcriptional activationdomain (or functional fragment thereof), the ZFP DNA-binding domain andthe transcriptional activation domain (or functional fragment thereof)are in operative linkage if, in the fusion polypeptide, the ZFPDNA-binding domain portion is able to bind its target site and/or itsbinding site, while the transcriptional activation domain (or functionalfragment thereof) is able to activate transcription.

[0044] “Specific binding” between, for example, a ZFP and a specifictarget site means a binding affinity of at least 1×10⁶ M⁻¹.

[0045] A “regulatory domain” or “functional domain” refers to apolypeptide, protein or protein domain that has transcriptionalmodulation activity when tethered to a DNA binding domain, e.g., a ZFP.Typically, a regulatory domain is covalently or non-covalently linked toa ZFP (e.g., to form a fusion molecule) to effect transcriptionmodulation. Regulatory domains can be activation domains or repressiondomains. Activation domains include, but are not limited to, VP16, VP64,ligand-bound wild-type thyroid hormone receptor (TR) and certain TRmutants, and the p65 subunit of nuclear factor Kappa-B. Repressiondomains include, but are not limited to, KRAB, MBD2B, unligandedwild-type TR and certain TR mutants, and v-ErbA. Additional regulatorydomains include, e.g., transcription factors and co-factors (e.g., MAD,ERD, SID, early growth response factor 1, and nuclear hormonereceptors), endonucleases, integrases, recombinases, methyltransferases,histone acetyltransferases, histone deacetylases etc. Activators andrepressors include co-activators and co-repressors (see, e.g., Utley etal., Nature 394:498-502 (1998)). Alternatively, a ZFP can act alone,without a regulatory domain, to effect transcription modulation.

[0046] A “fusion molecule” is a molecule in which two or more subunitmolecules are linked, preferably covalently. The subunit molecules canbe the same chemical type of molecule, or can be different chemicaltypes of molecules. Examples of the first type of fusion moleculeinclude, but are not limited to, fusion polypeptides (for example, afusion between a ZFP DNA-binding domain and a methyl binding domain) andfusion nucleic acids (for example, a nucleic acid encoding the fusionpolypeptide described herein). Examples of the second type of fusionmolecule include, but are not limited to, a fusion between atriplex-forming nucleic acid and a polypeptide, and a fusion between aminor groove binder and a nucleic acid.

[0047] An “exogenous molecule” is a molecule that is not normallypresent in a cell, but can be introduced into a cell by one or moregenetic, biochemical or other methods. Normal presence in the cell isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

[0048] An exogenous molecule can be, among other things, a smallmolecule, such as is generated by a combinatorial chemistry process, ora macromolecule such as a protein, nucleic acid, carbohydrate, lipid,glycoprotein, lipoprotien, polysaccharide, any modified derivative ofthe above molecules, or any complex comprising one or more of the abovemolecules. Nucleic acids include DNA and RNA, can be single- ordouble-stranded; can be linear, branched or circular; and can be of anylength. Nucleic acids include those capable of forming duplexes, as wellas triplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

[0049] An exogenous molecule can be the same type of molecule as anendogenous molecule, e.g., protein or nucleic acid (i.e., an exogenousgene), providing it has a sequence that is different from an endogenousmolecule. For example, an exogenous nucleic acid can comprise a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

[0050] By contrast, an “endogenous molecule” is one that is normallypresent in a particular cell at a particular developmental stage underparticular environmental conditions. For example, an endogenous nucleicacid can comprise a chromosome, the genome of a mitochondrion,chloroplast or other organelle, or a naturally-occurring episomalnucleic acid. Additional endogenous molecules can include endogenousgenes and endogenous proteins, for example, transcription factors andcomponents of chromatin remodeling complexes.

[0051] A “gene,” for the purposes of the present disclosure, includes aDNA region encoding a gene product (see below), as well as all DNAregions which regulate the production of the gene product, whether ornot such regulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions. A gene canbe an endogenous cellular gene, either normal or mutant, or an exogenousgene of an infecting organism such as, for example, a bacterium orvirus.

[0052] An “endogenous cellular gene” refers to a gene which is native toa cell, which is in its normal genomic and chromatin context, and whichis not heterologous to the cell. Such cellular genes include, e.g.,animal genes, plant genes, bacterial genes, protozoal genes fungalgenes, mitochondrial genes and chloroplastic genes.

[0053] A “native chromatin context” refers to the naturally occurring,structural relationship of genomic DNA (e.g., bacterial, animal, fungal,plant, protozoal, mitochondrial, and chloroplastic) and DNA-bindingproteins (e.g., histones, non-histone chromosomal proteins and bacterialDNA binding protein II), which together form chromosomes. An endogenouscellular gene can be in a transcriptionally active or inactive state inits native chromatin context.

[0054] “Gene expression” refers to the conversion of the information,contained in a gene, into a gene product. A gene product can be thedirect transcriptional product of a gene (e.g., mRNA, tRNA, rRNA,antisense RNA, ribozyme, structural RNA or any other type of RNA) or aprotein produced by translation of a mRNA. Gene products also includeRNAs which are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

[0055] “Gene activation” and “augmentation of gene expression” refer toany process which results in an increase in production of a geneproduct. A gene product can be either RNA (including, but not limitedto, mRNA, rRNA, tRNA, and structural RNA) or protein. Accordingly, geneactivation includes those processes which increase transcription of agene and/or translation of a mRNA. Examples of gene activation processeswhich increase transcription include, but are not limited to, thosewhich facilitate formation of a transcription initiation complex, thosewhich increase transcription initiation rate, those which increasetranscription elongation rate, those which increase processivity oftranscription and those which relieve transcriptional repression (by,for example, blocking the binding of a transcriptional repressor). Geneactivation can constitute, for example, inhibition of repression as wellas stimulation of expression above an existing level. Examples of geneactivation processes which increase translation include those whichincrease translational initiation, those which increase translationalelongation and those which increase mRNA stability. In general, geneactivation comprises any detectable increase in the production of a geneproduct, preferably an increase in production of a gene product by about2-fold, more preferably from about 2- to about 5-fold or any integralvalue therebetween, more preferably between about 5- and about 10-foldor any integral value therebetween, more preferably between about 10-and about 20-fold or any integral value therebetween, still morepreferably between about 20- and about 50-fold or any integral valuetherebetween, more preferably between about 50- and about 100-fold orany integral value therebetween, more preferably 100-fold or more.

[0056] “Gene repression” and “inhibition of gene expression” refer toany process which results in a decrease in production of a gene product.A gene product can be either RNA (including, but not limited to, mRNA,rRNA, tRNA, and structural RNA) or protein. Accordingly, gene repressionincludes those processes which decrease transcription of a gene and/ortranslation of a mRNA. Examples of gene repression processes whichdecrease transcription include, but are not limited to, those whichinhibit formation of a transcription initiation complex, those whichdecrease transcription initiation rate, those which decreasetranscription elongation rate, those which decrease processivity oftranscription and those which antagonize transcriptional activation (by,for example, blocking the binding of a transcriptional activator). Generepression can constitute, for example, prevention of activation as wellas inhibition of expression below an existing level. Examples of generepression processes which decrease translation include those whichdecrease translational initiation, those which decrease translationalelongation and those which decrease mRNA stability. Transcriptionalrepression includes both reversible and irreversible inactivation ofgene transcription. In general, gene repression comprises any detectabledecrease in the production of a gene product, preferably a decrease inproduction of a gene product by about 2-fold, more preferably from about2- to about 5-fold or any integral value therebetween, more preferablybetween about 5- and about 10-fold or any integral value therebetween,more preferably between about 10- and about 20-fold or any integralvalue therebetween, still more preferably between about 20- and about50-fold or any integral value therebetween, more preferably betweenabout 50- and about 100-fold or any integral value therebetween, morepreferably 100-fold or more. Most preferably, gene repression results incomplete inhibition of gene expression, such that no gene product isdetectable.

[0057] The term “modulate” refers to the suppression, enhancement orinduction of a function. For example, a ZFP can modulate gene expressionby binding to a motif within a transcriptional control sequence, therebyenhancing or suppressing transcription of a gene operatively linked tothe transcriptional control sequence. Additionally, modulation includesinhibition of transcription of a gene by virtue of a ZFP binding to agene and blocking DNA dependent RNA polymerase from reading through thegene, thus inhibiting transcription of the gene. Furthermore, modulationincludes inhibition of translation of a transcript. Thus, “modulation”of gene expression includes both gene activation and gene repression.

[0058] Modulation can be assayed by determining any parameter that isindirectly or directly affected by the expression of the target gene.Such parameters include, e.g., changes in RNA or protein levels; changesin protein activity; changes in product levels; changes in downstreamgene expression; changes in transcription or activity of reporter genessuch as, for example, luciferase, CAT, beta-galactosidase,beta-glucuronidase, horseradish peroxidase, alkaline phosphatase, GFP(see, e.g., Mistili & Spector, (1997) Nature Biotechnology 15:961-964)or selectable markers such as drug resistance; changes in signaltransduction; changes in phosphorylation and dephosphorylation; changesin receptor-ligand interactions; changes in concentrations of secondmessengers such as, for example, cGMP, cAMP, IP₃, and Ca2⁺; changes incell growth, changes in vascularization, and/or changes in anyfunctional effect of gene expression. Measurements can be made in vitro,in vivo, and/or ex vivo. Such functional effects can be measured byconventional methods, e.g., measurement of RNA or protein levels,measurement of RNA stability, and/or identification of downstream orreporter gene expression. Readout can be by way of, for example,chemiluminescence, fluorescence, calorimetric reactions, antibodybinding, inducible markers, ligand binding assays; changes inintracellular second messengers such as cGMP and inositol triphosphate(IP₃); changes in intracellular calcium levels; cytokine release, andthe like.

[0059] When a zinc finger protein (or fusion molecule) is used toinhibit the expression of a cellular protein, the expression level ofthe cellular protein, and/or the mRNA encoding it, is preferably lessthan 75% of that in a reference cell that does not express or otherwisecontain the zinc finger protein effecting regulation. Preferably, theexpression level is less than 50% of that in the reference cell; morepreferably it is less than 25% of that in the reference cell; mostpreferably it is less than 5% of that in the reference cell. When a zincfinger protein is used to activate the expression of a cellular protein,the expression level is more than 110% of that in a reference cell notexpressing the ZFP. Preferably, the expression level is more than 125%of that in the reference cell; more preferably it is more than 150% ofthat in the original cell; most preferably it is more than 175% of thatin the reference cell.

[0060] The terms “transcriptional control element,” “transcriptionalregulatory element,” “transcriptional control sequence” and“transcriptional regulatory sequence” are used interchangeably to referto DNA sequences which mediate modulation of transcription. Examples ofsuch elements or sequences include, but are not limited to, promoters,operators, enhancers, silencers, splice donor and acceptor sites,transcription termination sites and polyadenylation sites. Atranscriptional control sequence is responsive to a molecular target ifthe molecular target participates, either directly or indirectly, inmodulation of transcription mediated by the control element.

[0061] “Eukaryotic cells” include, but are not limited to, fungal cells(such as yeast), protozoal cells, archael cells, plant cells, animalcells, mammalian cells and human cells. Similarly, “prokaryotic cells”include, but are not limited to, bacteria.

[0062] A “functional fragment” of a protein, polypeptide or nucleic acidis a protein, polypeptide or nucleic acid whose sequence is notidentical to the full-length protein, polypeptide or nucleic acid, yetretains one or more functions exhibited by the full-length protein,polypeptide or nucleic acid. A functional fragment can possess more,fewer, or the same number of residues as the corresponding nativemolecule, and/or can contain one or more amino acid or nucleotidesubstitutions. Methods for determining the function of a nucleic acid(e.g., coding function, ability to hybridize to another nucleic acid)are well-known in the art. Similarly, methods for determining proteinfunction are well-known. For example, the DNA-binding function of apolypeptide can be determined, for example, by filter-binding,electrophoretic mobility-shift, or immunoprecipitation assays. SeeAusubel et al., supra. The ability of a protein to interact with anotherprotein can be determined, for example, by co-immunoprecipitation,two-hybrid assays or complementation, both genetic and biochemical. See,for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

[0063] A “target site” or “target sequence” is a sequence that is boundby a binding protein such as, for example, a ZFP. Target sequences canbe nucleotide sequences (either DNA or RNA) or amino acid sequences. Byway of example, a DNA target sequence for a three-finger ZFP isgenerally either 9 or 10 nucleotides in length, depending upon thepresence and/or nature of cross-strand interactions between the ZFP andthe target sequence.

[0064] A “target subsite” or “subsite” is the portion of a DNA targetsite that is bound by a single zinc finger. Thus, in the absence ofcross-strand interactions, a subsite is generally three nucleotides inlength. In cases in which a cross-strand interaction occurs (e.g., a“D-able subsite” as described, for example, in co-owned PCT WO 00/42219,incorporated by reference in its entirety herein), a subsite is fournucleotides in length and overlaps with another 3- or 4-nucleotidesubsite.

[0065] A “molecular target” refers to any molecule, for example amolecule within a cell or associated with a cell membrane, that is beingexamined for interaction with a candidate compound (e.g., a drug).Non-limiting examples of molecular targets include DNA, RNA and proteinssuch as receptors (e.g., cell surface, membrane-bound or nuclear),components of signal transduction pathways, transcription factors orfunctional fragments thereof. A molecular targets can also comprise amacromolecule such as a protein, nucleic acid, carbohydrate, lipid,glycoprotein, lipoprotien, polysaccharide, any modified derivative ofthe above molecules, or any complex comprising one or more of the abovemolecules. A compound “interacts” with a molecular target when itaffects, directly or indirectly, the molecular target. The compound canact directly on the molecular target, for example when the moleculartarget is a protein, the compound may directly interact with the proteinby binding to it or may directly regulate expression of the protein viaaction on transcriptional regulatory elements. Similarly, the compoundmay also act indirectly on the molecular target, for example by blockingor stimulating a separate molecule that in turn acts on the moleculartarget. Indirect action of a compound on a molecular target can occur,for example, when the target is a non-protein molecule and the compoundinteracts with a protein involved in the production, stability,activity, maintenance and/or modification of the non-protein moleculartarget.

[0066] The term “effective amount” includes that amount which results inthe desired result, for example, deactivation of a previously activatedgene or activation of an inactive gene, or that amount which results inthe inactivation of a gene containing a zinc finger-nucleotide bindingmotif, or that amount which blocks transcription of a structural gene ortranslation of RNA.

[0067] “K_(d)” refers to the dissociation constant for the compound,i.e., the concentration of a compound (e.g., a zinc finger protein) thatgives half maximal binding of the compound to its target (i.e., half ofthe compound molecules are bound to the target) under given conditions(i.e., when [target]<<K_(d)), as measured using a given assay system(see, e.g., U.S. Pat. No. 5,789,538). The assay system used to measurethe K_(d) should be chosen so that it gives the most accurate measure ofthe actual K_(d) of the ZFP. Any assay system can be used, as long is itgives an accurate measurement of the actual K_(d) of the ZFP. In oneembodiment, the K_(d) for the ZFPs is measured using an electrophoreticmobility shift assay (“EMSA”), as described in Example 1 of the presentspecification. Unless an adjustment is made for ZFP purity or activity,the K_(d) calculations made using the method of Example 1 may result inan underestimate of the true K_(d) of a given ZFP. Preferably, the K_(d)of a ZFP used to modulate transcription of an endogenous cellular geneis less than about 100 nM, more preferably less than about 75 nM, morepreferably less than about 50 nM, most preferably less than about 25 nM.

[0068] Screening Assays

[0069] In preferred embodiments, screening assays are conducted usingcells comprising at least one exogenous zinc finger protein or usingmaterials derived from cells comprising at least one exogenous zincfinger protein. The screening assays described herein allow for highthroughput screening of candidate compounds, which can be accomplishedwhile reducing the incidence of false positives.

[0070] A. Zinc Finger Proteins

[0071] The compositions and methods disclosed herein involve use of DNAbinding proteins, particularly zinc finger proteins. See, for example,Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes et al. (1993)Scientific American Feb.:56-65; and Klug (1999) J. Mol. Biol.293:215-218. The three-fingered Zif268 murine transcription factor hasbeen particularly well studied. (Pavletich, N. P. & Pabo, C. O. (1991)Science 252:809-17). The X-ray co-crystal structure of Zif268 ZFP anddouble-stranded DNA indicates that each finger interacts independentlywith DNA (Nolte et al. (1998) Proc Natl Acad Sci USA 95:2938-43;Pavletich, N. P. & Pabo, C. O. (1993) Science 261:1701-7). Theorganization of the 3-fingered domain allows recognition of threecontiguous base-pair triplets by each finger. Each finger isapproximately 30 amino acids long, adopting a ββα fold. The twoβ-strands form a sheet, positioning the recognition α-helix in the majorgroove for DNA binding. Specific contacts with the bases are mediatedprimarily by four amino acids immediately preceding and within therecognition helix. Conventionally, these recognition residues arenumbered −1, 2, 3, and 6 based on their positions in the α-helix.

[0072] ZFP DNA-binding domains are designed and/or selected to recognizea particular target site as described in co-owned WO 00/42219; WO00/41566; and U.S. Ser. Nos. 09/444,241 filed Nov. 19, 1999; 09/535,088filed Mar. 23, 2000; as well as U.S. Pat. Nos. 5,789,538; 6,007,408;6,013,453; 6,140,081; and 6,140,466; and PCT publications WO 95/19431,WO 98/54311, WO 00/23464 and WO 00/27878. In one embodiment, a targetsite for a zinc finger DNA-binding domain is identified according tosite selection rules disclosed in co-owned WO 00/42219. In a preferredembodiment, a ZFP is designed and optimized as described in co-ownedU.S. Ser. No. 09/716,637, filed Nov. 20, 2000, entitled “IterativeOptimization in the Design of Binding Proteins.” In certain preferredembodiments, the binding specificity of the DNA-binding domain isdirected to one or more accessible regions in the sequence in question(e.g., in cellular chromatin). Accessible regions are determined asdescribed, for example, in co-owned U.S. patent application Ser. Nos.entitled “Methods for Binding an Exogenous Molecule to CellularChromatin,” Ser. No. 60/200,590, filed Apr. 28, 2000 and “Databases ofRegulatory Sequences; Methods of Making and Using Same,” Ser. No.60/228,556, filed Aug. 28, 2000, the disclosures of which are herebyincorporated by reference herein. A DNA-binding domain is then designedand/or selected and/or optimized to bind to a target site within theaccessible region.

[0073] Two alternative methods are typically used to create the codingsequences required to express newly designed DNA-binding peptides. Anexample of one protocol is a PCR-based assembly procedure, forconstruction of a three-finger ZFP, that utilizes six overlappingoligonucleotides (see FIG. 1). Three oligonucleotides correspond to“universal” sequences that encode portions of the DNA-binding domainbetween the recognition helices. These oligonucleotides remain constantfor all zinc finger constructs. The other three “specific”oligonucleotides are designed to encode the recognition helices. Theseoligonucleotides contain substitutions primarily at positions −1, 2, 3and 6 on the recognition helices such that each specifies a distinctDNA-binding domain.

[0074] The PCR synthesis is carried out in two steps. First, a doublestranded DNA template is created by combining the six oligonucleotides(three universal, three specific) in a four cycle PCR reaction with alow temperature annealing step, thereby annealing the oligonucleotidesto form a DNA “scaffold.” The gaps in the scaffold are filled in by ahigh-fidelity thermostable polymerase, the combination of Taq and Pfupolymerases also suffices. In the second phase of construction, the zincfinger template is amplified using external primers designed toincorporate restriction sites at both ends of the amplification product,to facilitate its cloning into a shuttle vector or directly into anexpression vector.

[0075] An alternative method of assembling a sequence to encode adesigned DNA-binding protein relies on annealing complementaryoligonucleotides encoding the specific regions of the desired zincfinger protein. This particular application requires that theoligonucleotides be phosphorylated prior to the final ligation step.This is usually performed before setting up the annealing reactions, butphosphorylation (e.g., by polynucleotide kinase) can also occurpost-annealing. In brief, the “universal” oligonucleotides encoding theconstant regions of the proteins are annealed to complementaryoligonucleotides. Additionally, the “specific” oligonucleotides encodingthe finger recognition helices are annealed with their respectivecomplementary oligonucleotides. These complementary oligos are designedto fill in the region, which was previously filled in by polymerase inthe protocol described above. Oligonucleotides complementary touniversal oligo 1 and specific oligo 6 (encoding finger 3) areengineered to contain overhanging sequences specific for the restrictionsites used in cloning into the vector of choice. The second assemblyprotocol differs from the initial protocol in the following aspects: the“scaffold” encoding the newly designed zinc finger protein is composedentirely of synthetic DNA, thereby eliminating the polymerase fill-instep, additionally the fragment to be cloned into the vector does notrequire amplification. Lastly, the inclusion of sequence-specificoverhangs eliminates the need for restriction enzyme digestion prior toinsertion into a vector.

[0076] The resulting fragment encoding the newly designed zinc fingerprotein is ligated into an expression vector. Expression vectors thatare commonly utilized include, but are not limited to, a modifiedpMAL-c2 bacterial expression vector (New England BioLabs, “NEB,”Beverly, Mass.) or a eukaryotic expression vector, pcDNA (Promega,Madison, Wis.). Conventional methods of purification can be used (seeAusubel, supra, Sambrook, supra). In addition, any suitable host can beused, e.g., bacterial cells, insect cells, yeast cells, mammalian cells,and the like.

[0077] Expression of the zinc finger protein fused to a maltose bindingprotein (MBP-ZFP) in bacterial strain JM109 allows for straightforwardpurification through an amylose column (NEB). High expression levels ofthe zinc finger chimeric protein can be obtained by induction with IPTGsince the MBP-ZFP fusion in the pMal-c2 expression plasmid is under thecontrol of the IPTG inducible tac promoter (NEB). Bacteria containingthe MBP-ZFP fusion plasmids are inoculated in to 2×YT medium containing10 μM ZnCl₂, 0.02% glucose, plus 50 μg/ml ampicillin and shaken at 37°C. At mid-exponential growth IPTG is added to 0.3 mM and the culturesare allowed to shake. After 3 hours the bacteria are harvested bycentrifugation, disrupted by sonication, and then insoluble material isremoved by centrifugation. The MBP-ZFP proteins are captured on anamylose-bound resin, washed extensively with buffer containing 20 mMTris-HCl (pH 7.5), 200 mM NaCl, 5 mM DTT and 50 μM ZnCl₂, then elutedwith maltose in essentially the same buffer (purification is based on astandard protocol from NEB). Purified proteins are quantitated andstored for biochemical analysis.

[0078] The biochemical properties of the purified proteins, e.g., K_(d),can be characterized by any suitable assay. K_(d) can be characterizedvia electrophoretic mobility shift assays (“EMSA”) (Buratowski &Chodosh, in Current Protocols in Molecular Biology pp. 12.2.1-12.2.7(Ausubel ed., 1996); see also U.S. Pat. No. 5,789,538; PCT WO 00/42219,hereby incorporated by reference). Affinity is measured by titratingpurified protein against a low fixed amount of labeled double-strandedoligonucleotide target. The target comprises the natural binding sitesequence (9 or 18 bp) flanked by the 3 bp found in the natural sequence.External to the binding site plus flanking sequence is a constantsequence. The annealed oligonucleotide targets possess a 1 bp 5′overhang which allows for efficient labeling of the target with T4 phagepolynucleotide kinase. For the assay the target is added at aconcentration of 40 nM or lower (the actual concentration is kept atleast 10-fold lower than the lowest protein dilution) and the reactionis allowed to equilibrate for at least 45 min. In addition the reactionmixture also contains 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 0.1mM ZnCl₂, 5 mM DTT, 10% glycerol, 0.02% BSA (poly (dIdC) or (dAdT)(Pharmacia) can also added at 10-100 μg/μl).

[0079] The equilibrated reactions are loaded onto a 10% polyacrylamidegel, which has been pre-run for 45 min in Tris/glycine buffer, thenbound and unbound labeled target is resolved be electrophoresis at 150V(alternatively, 10-20% gradient Tris-HCl gels, containing a 4%polyacrylamide stacker, can be used). The dried gels are visualized byautoradiography or phosphoroimaging and the apparent K_(d) is determinedby calculating the protein concentration that gives half-maximalbinding.

[0080] Similar assays can also include determining active fractions inthe protein preparations. Active fractions are determined bystoichiometric gel shifts where proteins are titrated against a highconcentration of target DNA. Titrations are done at 100, 50, and 25% oftarget (usually at micromolar levels).

[0081] B. Fusion Molecules

[0082] The selection and/or design of zinc finger-containing proteinsalso allows for the design of fusion molecules which facilitateregulation of gene expression. Thus, in certain embodiments, thecompositions and methods disclosed herein involve fusions between a zincfinger protein (or functional fragment thereof) and one or morefunctional domains (or functional fragments thereof), or apolynucleotide encoding such a fusion. When such a fusion molecule ispresent in a cell, a functional domain is brought into proximity with asequence in a gene that is bound by the zinc finger protein. Thetranscriptional regulatory function of the functional domain is thenable to act on the gene, by, for example, modulating expression of thegene.

[0083] The zinc finger protein can be covalently or non-covalentlyassociated with one or more regulatory domains, alternatively two ormore regulatory domains, with the two or more domains being two copiesof the same domain, or two different domains. The regulatory domains canbe covalently linked to the zinc finger protein, e.g., via an amino acidlinker, as part of a fusion protein. The zinc finger proteins can alsobe associated with a regulatory domain via a non-covalent dimerizationdomain, e.g., a leucine zipper, a STAT protein N terminal domain, or anFK506 binding protein (see, e.g., O'Shea, Science 254: 539 (1991),Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211:121-128(1996); Klemm et al., Annu. Rev. Immunol. 16:569-592 (1998); Klemm etal., Annu. Rev. Immunol. 16:569-592 (1998); Ho et al., Nature382:822-826 (1996); and Pomeranz et al., Biochem. 37:965 (1998)). Theregulatory domain can be associated with the zinc finger protein at anysuitable position, including the C- or N-terminus of the zinc fingerprotein.

[0084] Common regulatory domains for addition to the zinc finger proteininclude, e.g., effector domains from transcription factors (activators,repressors, co-activators, co-repressors), silencers, nuclear hormonereceptors, oncogene transcription factors (e.g., myc, jun, fos, myb,max, mad, rel, ets, bcl, myb, mos and/or erb family members etc.); DNArepair enzymes and their associated factors and modifiers; DNArearrangement enzymes and their associated factors and modifiers;chromatin associated proteins and their modifiers (e.g., kinases,acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers.

[0085] Transcription factor polypeptides from which one can obtain aregulatory domain include those that are involved in regulated and basaltranscription. Such polypeptides include, for example, transcriptionfactors, their effector domains, coactivators, silencers, and nuclearhormone receptors. See, e.g., Goodrich et al., Cell 84:825-30 (1996) fora review of proteins and nucleic acid elements involved intranscription; transcription factors in general are reviewed in Barnes &Adcock, Clin. Exp. Allergy 25 Suppl. 2:46-9 (1995) and Roeder, MethodsEnzymol. 273:165-71 (1996). Databases dedicated to transcription factorsare known (see, e.g., Science 269:630 (1995)). Nuclear hormone receptortranscription factors are described in, for example, Rosen et al., J.Med. Chem. 38:4855-74 (1995). The C/EBP family of transcription factorsare reviewed in Wedel et al., Immunobiology 193:171-85 (1995).Coactivators and co-repressors that mediate transcription regulation bynuclear hormone receptors are reviewed in, for example, Meier, Eur. J.Endocrinol. 134(2):158-9 (1996); Kaiser et al., Trends Biochem. Sci.21:342-5 (1996); and Utley et al., Nature 394:498-502 (1998)). GATAtranscription factors, which are involved in regulation ofhematopoiesis, are described in, for example, Simon, Nat. Genet. 11:9-11(1995); Weiss et al., Exp. Hematol. 23:99-107. TATA box binding protein(TBP) and its associated TAF polypeptides (which include TAF30, TAF55,TAF80, TAF110, TAF150, and TAF250) are described in Goodrich & Tjian,Curr. Opin. Cell Biol. 6:403-9 (1994) and Hurley, Curr. Opin. Struct.Biol. 6:69-75 (1996). The STAT family of transcription factors isreviewed in, for example, Barahmand-Pour et al., Curr. Top. Microbiol.Immunol. 211:121-8 (1996). Transcription factors involved in disease arereviewed in Aso et al., J. Clin. Invest. 97:1561-9 (1996).

[0086] An exemplary functional domain for fusing with a ZFP is a KRABrepression domain from the human KOX-1 protein (see, e.g., Thiesen etal., New Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad.Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res.22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91,4514-4518 (1994). Another suitable repression domain is methyl bindingdomain protein 2B (MBD-2B) (see, also Hendrich et al. (1999) Mamm Genome10:906-912 for description of MBD proteins). Another useful repressiondomain is that associated with the v-ErbA protein. See, for example,Damm, et al. (1989) Nature 339:593-597; Evans (1989) Int. J. CancerSuppl. 4:26-28; Pain et al. (1990) New Biol. 2:284-294; Sap et al.(1989) Nature 340:242-244; Zenke et al. (1988) Cell 52:107-119; andZenke et al. (1990) Cell 61:1035-1049. Additional exemplary repressiondomains include, but are not limited to, unliganded (e.g., not bound toT3) thyroid hormone receptor (TR) and certain TR mutants, SID, MBD2,MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, andMeCP2. See, for example, Zhang et al. (2000); Ann Rev Physiol62:439-466; Bird et al. (1999) Cell 99:451-454; Tyler et al. (1999) Cell99:443-446; Knoepfler et al. (1999) Cell 99:447-450; and Robertson etal. (2000) Nature Genet. 25:338-342. Additional exemplary repressiondomains include, but are not limited to, ROM2 and AtHD2A. See, forexample, Chern et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000)Plant J. 22:19-27.

[0087] Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); ligand-bound TR and certain TRmutants; the p65 subunit of nuclear factor kappa B (Bitko & Barik, J.Virol. 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942(1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificialchimeric functional domains such as VP64 (Seifpal et al., EMBO J. 11,4961-4968 (1992)).

[0088] Additional exemplary activation domains include, but are notlimited to, p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, forexample, Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood etal. (1999) J. Mol. Endocrinol. 23:255-275; Leo et al. (2000) Gene245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89;McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik etal. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999)Curr. Opin. Genet. Dev. 9:499-504. Additional exemplary activationdomains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5,-6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP, and TRAB1. See, for example,Ogawa et al. (2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells1:87-99; Goff et al. (1991) Genes Dev. 5:298-309; Cho et al. (1999)Plant Mol. Biol. 40:419-429; Ulmason et al. (1999) Proc. Natl. Acad.Sci. USA 96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22:1-8;Gong et al. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999)Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

[0089] Additional functional domains are disclosed, for example, inco-owned WO 00/41566. Further, insulator domains, chromatin remodelingproteins such as ISWI-containing domains and/or methyl binding domainproteins suitable for use in fusion molecules are described, forexample, in co-owned U.S. patent applications Ser. No. 60/236,409,entitled “Nuclear Reprogramming Using ISWI And Related ChromatinRemodeling ATPases”; Ser. No. 60/236,884, entitled “Modulation Of GeneExpression Using Methyl Binding Domain Polypeptides;” and Ser. No.60/253,678, entitled “Modulation of Gene Expression Using InsulatorBinding Proteins.”

[0090] Functional domains can also be derived from nuclear hormonereceptors. For example, the thyroid hormone receptor (TR) is a member ofthe nuclear hormone receptor superfamily and is normally boundconstitutively to its target genes. The effect of TR binding (i.e.,either repression or activation of gene expression) ordinarily dependsupon the presence or absence of its ligand, thyroid hormone (T3). In theabsence of T3 the receptor generally represses gene expression to alevel below the basal level. A number of proteins have been identifiedthat are recruited by the unliganded receptor and are believed toconstitute a repressive complex. Examples of such proteins include SMRTand NCoR, which interact directly with the receptor, as well as Sin3,which interacts with SMRT/NCoR. Sin3 also interacts with a number ofhistone deacetylases, for example, HDACs 1 through 8 (some of which mayalso interact directly with TR). Recruitment of histone deacetylases byDNA-bound TR is believed to play a major role in its ability to conferrepression; however, it is also possible that repressive factors otherthan HDACs are recruited by TR.

[0091] Binding of ligand to DNA-bound TR results in the decay of therepressive complex associated with the TR and recruitment of activatingfactors to the DNA-bound, ligand-bound TR. Such activating factorsinclude, but are not limited to, the histone acetyltransferases SRC-1,CBP/p300 and P/CAF. Oligomeric activation complexes can also berecruited by ligand-bound TR, such as, for example, DRIP and ARC. Rachezet al. (1999) Nature 398:824-827; and Naar et al. (1999) Nature398:828-832. These have been shown to interact with other nuclearhormone receptors, in response to ligand binding, and facilitateactivation of gene expression in the context of a chromatin template.Another member of the nuclear receptor family, the glucocorticoidreceptor (GR), recruits the hBRG1/BAF chromatin remodeling complex inresponse to ligand binding. Fryer et al. (1998) Nature 393:88-91.

[0092] TR and related nuclear receptors are modular proteins comprisingan amino-terminal region (of undefined function), a central DNA bindingdomain and a carboxy-terminal ligand binding domain (LBD). The LBD, inaddition to binding hormone, is responsible for interactions with boththe repressive and activating factors described above. When the LBD isfused to a heterologous DNA binding domain (Gal4), it mediatesrepression of a target promoter containing a Gal4 binding site.Collingwood et al. (1998) EMBO J. 17:4760-4770. In addition,T3-dependent activation of transcription can be achieved using a fusionof the TR LBD with the Gal4 DNA-binding domain. Tone et al. (1994) J.Biol. Chem. 269:31,157-31,161.

[0093] Knowledge of the structure of the LBD of TR and related nuclearreceptors, together with the results of mutagenesis studies, can be usedto design mutant receptors whose repression and activation activity areimpervious to hormone concentration. For example, single amino acidmutants of TR that are unable to bind physiological levels of T3 (e.g.G344E, Δ430M, and Δ276I) recruit corepressors to their binding site.Collingwood et al. (1994) Mol. Endocrinol. 8:1262-1277; Collingwood etal. (1998) supra. Conversely, mutations causing conformational changesin the ligand binding domain that mimic those induced by hormone bindinghave been identified in the estrogen receptor (e.g. L536P and Y541D/E/A)and generate constitutively activating forms of the receptor. Eng et al.(1997) Mol. Cell. Biol. 17:4644-4653; White et al. (1997) EMBO J.16:1427-1435.

[0094] Accordingly, a mutant nuclear hormone receptor LBD derived, forexample, from TR or GR can be used as a component of a fusion with a ZFPDNA-binding domain, to recruit activating or repressing proteincomplexes to a region of interest in cellular chromatin, therebyregulating expression of a target molecule. Certain naturally-occurringmutant LBDs are available; and new mutants can be constructed by methodswell-known to those of skill in the art. The site of action of suchcomplexes is determined by the specificity of the DNA-binding domain;while their activity is determined by the nature of the mutation to theLBD and is independent of ligand concentration. For instance, a fusioncomprising a LBD that has been mutated such that it is unable to bindhormone will facilitate formation of repressive complexes; while afusion molecule comprising a LBD mutation that changes the conformationof the LBD such that it resembles a ligand-bound LBD will stimulate theformation of complexes that facilitate transcriptional activation. Thus,for the purposes of the present disclosure, a mutant nuclear hormonereceptor LBD (such as TR) can be used as an activation or repressiondomain.

[0095] In additional embodiments, targeted remodeling of chromatin, asdisclosed in co-owned U.S. patent application entitled “TargetedModification of Chromatin Structure,” can be used to generate one ormore sites in cellular chromatin that are accessible to the binding of afunctional domain/DNA binding domain fusion molecule.

[0096] Kinases, phosphatases, and other proteins that modifypolypeptides involved in gene regulation are also useful as functionaldomains for zinc finger proteins. Such modifiers are often involved inswitching on or off transcription mediated by, for example, hormones.Kinases involved in transcription regulation are reviewed in Davis, Mol.Reprod. Dev. 42:459-67 (1995), Jackson et al., Adv. Second MessengerPhosphoprotein Res. 28:279-86 (1993), and Boulikas, Crit. Rev. Eukaryot.Gene Expr. 5:1-77 (1995), while phosphatases are reviewed in, forexample, Schonthal & Semin, Cancer Biol. 6:239-48 (1995). Nucleartyrosine kinases are described in Wang, Trends Biochem. Sci. 19:373-6(1994).

[0097] Useful domains can also be obtained from the gene products ofoncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mosand/or erb family members) and their associated factors and modifiers.Oncogenes are described in, for example, Cooper, Oncogenes, The Jonesand Bartlett Series in Biology (2^(nd) ed., 1995). The ets transcriptionfactors are reviewed in Waslylk et al., Eur. J. Biochem. 211:7-18 (1993)and Crepieux et al., Crit. Rev. Oncog. 5:615-38 (1994). Myc oncogenesare reviewed in, for example, Ryan et al., Biochem. J. 314:713-21(1996). The jun and fos transcription factors are described in, forexample, The Fos and Jun Families of Transcription Factors (Angel &Herrlich, eds. 1994). The max oncogene is reviewed in Hurlin et al.,Cold Spring Harb. Symp. Quant. Biol. 59:109-16. The myb gene family isreviewed in Kanei-Ishii et al., Curr. Top. Microbiol. Immunol. 211:89-98(1996). The mos family is reviewed in Yew et al., Curr. Opin. Genet.Dev. 3:19-25 (1993).

[0098] Zinc finger proteins can include functional domains obtained fromDNA repair enzymes and their associated factors and modifiers. DNArepair systems are reviewed in, for example, Vos, Curr. Opin. Cell Biol.4:385-95 (1992); Sancar, Ann. Rev. Genet. 29:69-105 (1995); Lehmann,Genet. Eng. 17:1-19 (1995); and Wood, Ann. Rev. Biochem. 65:135-67(1996). DNA rearrangement enzymes and their associated factors andmodifiers can also be used as regulatory domains (see, e.g., Gangloff etal., Experientia 50:261-9 (1994); Sadowski, FASEB J. 7:760-7 (1993)).

[0099] Similarly, regulatory domains can be derived from DNA modifyingenzymes (e.g., DNA methyltransferases, topoisomerases, helicases,ligases, kinases, phosphatases, polymerases) and their associatedfactors and modifiers. Helicases are reviewed in Matson et al.,Bioessays, 16:13-22 (1994), and methyltransferases are described inCheng, Curr. Opin. Struct. Biol. 5:4-10 (1995). Chromatin associatedproteins and their modifiers (e.g., kinases, acetylases anddeacetylases), such as histone deacetylase (Wolffe, Science 272:371-2(1996)) are also useful as domains for addition to the zinc fingerprotein of choice. A DNA methyl transferase that acts as atranscriptional repressor can also be the regulatory domain. (see, e.g.,Van den Wyngaert et al., FEBS Lett. 426:283-289 (1998); Flynn et al., J.Mol. Biol. 279:101-116 (1998); Okano et al., Nucleic Acids Res.26:2536-2540 (1998); and Zardo & Caiafa, J. Biol. Chem. 273:16517-16520(1998)). Endonucleases such as Fok1 can also be used as transcriptionalrepressors, which act via gene cleavage (see, e.g., WO95/09233; andPCT/US94/01201).

[0100] Factors that control chromatin and DNA structure, movement andlocalization and their associated factors and modifiers; factors derivedfrom microbes (e.g., prokaryotes, eukaryotes and virus) and factors thatassociate with or modify them can also be used to obtain fusionproteins. Recombinases and integrases can be used as functional domains.Histone acetyltransferase can also be used as a transcriptionalactivator (see, e.g., Jin & Scotto, Mol. Cell. Biol. 18:4377-4384(1998); Wolffe, Science 272:371-372 (1996); Taunton et al., Science272:408-411 (1996); and Hassig et al., Proc. Natl. Acad. Sci. U.S.A.95:3519-3524 (1998)). Histone deacetylase can be used as atranscriptional repressor (see, e.g., Jin & Scotto, Mol. Cell. Biol.18:4377-4384 (1998); Syntichaki & Thireos, J. Biol. Chem.273:24414-24419 (1998); Sakaguchi et al., Genes Dev. 12:2831-2841(1998); and Martinez et al., J. Biol. Chem. 273:23781-23785 (1998)).

[0101] Linker domains between polypeptide domains, e.g., between twozinc finger proteins or between a zinc finger protein and a functionaldomain, can be included. Such linkers are typically polypeptidesequences, such as poly gly sequences of between about 5 and 200 aminoacids. Preferred linkers are typically flexible amino acid subsequenceswhich are synthesized as part of a recombinant fusion protein. Forexample, the linker DGGGS (SEQ ID NO: 28) can be used to link two zincfinger proteins. The flexible linker linking two zinc finger proteinscan also be an amino acid subsequence comprising the sequence TGEKP (SEQID NO: 29) (see, e.g., Liu et al., Proc. Natl. Acad. Sci. U.S.A.5525-5530 (1997)). The linker LRQKDGERP (SEQ ID NO: 30) can be used tolink two zinc finger proteins. The following linkers can also be used tolink two zinc finger proteins: GGRR (SEQ ID NO: 31) (Pomerantz et al.1995, supra), (G4S)_(n) (SEQ ID NO: 45) (Kim et al., Proc. Natl. Acad.Sci. U.S.A. 93, 1156-1160 (1996.); and GGRRGGGS (SEQ ID NO: 32);LRQRDGERP (SEQ ID NO: 33); LRQKDGGGSERP (SEQ ID NO: 34); LRQKd(G3S)₂ ERP(SEQ ID NO: 35). Alternatively, flexible linkers can be rationallydesigned using computer program capable of modeling both DNA-bindingsites and the peptides themselves (Desjarlais & Berg, Proc. Natl. Acad.Sci. U.S.A. 90:2256-2260 (1993), Proc. Natl. Acad. Sci. U.S.A.91:11099-11103 (1994) or by phage display methods (e.g., PCT WO99/45132).

[0102] A chemical linker can be used to connect synthetically orrecombinantly produced domain sequences. For example, poly(ethyleneglycol) linkers are available from Shearwater Polymers, Inc. Huntsville,Alabama. Some linkers have amide linkages, sulfhydryl linkages, orheterofunctional linkages. In addition to covalent linkage of zincfinger proteins to regulatory domains, non-covalent methods can be usedto produce molecules with zinc finger proteins associated withregulatory domains.

[0103] ZFP fusion molecules can also comprise, in addition to or insteadof one or more functional domains, one or more domains that facilitatepurification, expression monitoring and/or determination of cellularand/or subcellular localization. These include, for example,polypeptides such as maltose binding protein (“MBP”), glutathione Stransferase (GST), hexahistidine, c-myc, and the FLAG epitope.

[0104] Fusion molecules are constructed by methods of cloning andbiochemical conjugation that are well-known to those of skill in theart. In certain embodiments, fusion molecules comprise a zinc fingerprotein and at least two functional domains (e.g., an insulator domainor a methyl binding protein domain and, additionally, a transcriptionalactivation or repression domain). Fusion molecules also optionallycomprise nuclear localization signals (such as, for example, that fromthe SV40 medium T-antigen) and epitope tags (such as, for example, FLAGand hemagglutinin). Fusion proteins (and nucleic acids encoding them)are designed such that the translational reading frame is preservedamong the components of the fusion.

[0105] The fusion molecules disclosed herein comprise a zinc fingerbinding protein which binds to a target site and modulates expression ofa molecular target. In certain embodiments, the target site is presentin an accessible region of cellular chromatin. Accessible regions can bedetermined as described in co-owned U.S. patent applications Ser. No.60/200,590, Ser. No. 60/214,674 and Ser. No. 60/228,556. If the targetsite is not present in an accessible region of cellular chromatin, oneor more accessible regions can be generated as described in co-ownedU.S. patent application entitled “Targeted Modification of ChromatinStructure.” In additional embodiments, the zinc finger component of afusion molecule is capable of binding to cellular chromatin regardlessof whether its target site is in an accessible region or not. Forexample, such DNA binding domains are capable of binding to linker DNAand/or nucleosomal DNA. Examples of this type of “pioneer” DNA bindingdomain are found in certain steroid receptor and in hepatocyte nuclearfactor 3 (HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al.(1990) Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

[0106] Methods of gene regulation using one or more functional domains,targeted to a specific sequence by virtue of a fused DNA binding domain,can achieve modulation of gene expression. Modulation of gene expressioncan be in the form of decreased expression (e.g., repression). In thiscase, the value of a cellular property in a cell in which expression ofa molecular target is repressed by a ZFP is often lower than the valueof that property in a cell not expressing a repressive ZFP directed tothe molecular target. As described herein, repression of a specifictarget gene can be achieved by using a fusion molecule comprising a zincfinger protein and a functional domain.

[0107] Alternatively, modulation can be in the form of increased geneexpression, or activation, if activation of a gene encoding themolecular target is required to test interaction of a compound with amolecular target. The functional domain (e.g., insulator domain,activation domain, etc.) enables increased and/or sustained expressionof the target gene. The value of a cellular property in a cell in whichexpression of a molecular target is increased by a ZFP is often greaterthan the value of that property in a cell not expressing an activatingZFP directed to the molecular target.

[0108] Some cells are designed to express a sequence encoding a zincfinger protein in operable linkage to an inducible promoter. A varietyof inducible promoters are available; many of which can be regulated bysmall molecules or other environmental factors such as, for example,temperature and nutritional conditions. Operable linkage to an induciblepromoter allows activation of a ZFP, and thereby modulation ofexpression of a molecular target by the ZFP, to be controlled bysupplying the cell with the appropriate small molecule or inducingstimulus. Regulation of expression of ZFPs by inducible promoters isuseful for achieving transient modulation of expression of a moleculartarget whose permanent over- or under-expression would result inlethality to the cell. Inducible expression is also advantageous inreducing secondary effects due to modulation of a molecular target. Forexample, modulation of expression of one cellular protein can directlyor indirectly result in changes in the relative abundance of many othersproteins (and other molecules) within the cell. By inducing expressionof a zinc finger protein shortly before an assay is performed, suchsecondary changes are minimized. Accordingly, differences in responsebetween a test cell, comprising a regulated molecular target, and acontrol cell, are entirely or substantially entirely due to interactionbetween the compound and the molecular target rather than to secondaryeffects caused by regulation of the target.

[0109] C. Cell-Based Assays

[0110] In one aspect, methods of performing cell-based drug-screeningassays that reduce the incidence of false positives common to otherconventional methods are described. False positives occur, for example,when a compound does not interact with an endogenous target but achievesthe same or similar effect through an alternative mechanism.

[0111] In the present methods, false positives are reduced or eliminatedby performing assays on matched populations of cells that preferablydiffer only in the expression level of at least one molecular target(e.g., a protein target or a protein that modulates the expression ofthe molecular target). Typically, a control population of cellsexpresses a molecular target at levels that are normal for theparticular cell type and environmental (e.g., culture) conditions. Atest population of cells expresses a molecular target at altered levels(either higher or lower) compared to the control population. Alteredlevels of expression of the molecular target are achieved by a ZFP or aZFP fusion molecule which acts to modulate expression of the targetmolecule. Expression of the target molecule can be modulated directlyas, for example, when a ZFP or a ZFP fusion molecule regulatestranscription of a gene encoding the molecular target. Alternatively,indirect modulation of expression of a target molecule occurs when a ZFPor ZFP fusion molecule regulates expression of a gene that is involvedin the synthesis, stability or regulation of the target molecule.Indirect modulation can occur in cases in which the molecular target isnot a protein; although indirect modulation of a protein moleculartarget is also possible.

[0112] In one embodiment, a molecular target is overexpressed in thetest cells. A compound is screened in the test cell population for itsability to interact with the overexpressed molecular target, for exampleusing one or more assays that measure (quantitatively or qualitatively)interaction with the molecular target. The same compound is alsoscreened in the control population and its ability to interact with themolecular target is also determined using the assays used in the testcell population. If a compound elicits a similar response in these twopopulations of cells, it is likely that the compound is not exerting itseffect through an interaction with the molecular target. Conversely, ifa compound elicits different responses in these two populations ofcells, it is likely that the compound is interacting with the moleculartarget (e.g., directly or indirectly). Generally, if a molecular targetis overexpressed in a test cell, the magnitude of the response isgreater in the test cell than in the control cell. Thus, if a compoundsuspected of interacting with the target exerts a greater effect on thetest cell than on the control cell, the compound likely exerts itseffect through the intended target. However, in certain cases, themagnitude of a response can be smaller in a test cell that isoverexpressing a molecular target, for example, if the molecular targetrepresses a cellular component involved in the response.

[0113] In certain embodiments, the test and control cells differ in thatthe test cell does not express, or expresses at reduced levels, amolecular target for drug screening that is expressed in the controlcell. If a compound suspected of interacting with the target exerts asimilar effect on the control and test cells, then the compound is notexerting its effect through the intended molecular target interaction.For test cells which expresses a molecular target at levels lower thanthose at which it is expressed in a control cell, a compound thatinteracts with the molecular target will generally have a smaller effectin the test cells than in the control cells.

[0114] In other embodiments, the test and control cells differ in thatthe control cell does not express or expresses at reduced levels aprotein that is structurally similar to a molecular target and that cantransduce a similar cellular response to that resulting fromtransduction through the molecular target. If a compound suspected ofinteracting with the target exerts a different effect on the control andtest cells, then the compound is not exerting its effect through theintended molecular target interaction.

[0115] Test cells are preferably generated by regulation of cellulargenes with zinc finger proteins or fusion molecules described herein. Insome methods, a test cell is produced by engineering a cell to expressan exogenous zinc finger protein (or fusion molecule) designed torepress expression of an endogenous molecular target. In alternativemethods, a test cell is produced by engineering a cell to express anexogenous zinc finger protein (or fusion molecule) designed to activateor increase expression of an endogenous molecular target. The resultingtest cells are preferably substantially identical to the correspondingcontrol cells except for an exogenous nucleic acid encoding theexogenous zinc finger-containing molecule (and, possibly, a lowincidence of random mutations resulting from environmental factors).Thus, in certain embodiments, the phenotype of the test and controlscell populations will differ only in regard to the levels of theprotein(s) subject to regulation by the exogenous zinc finger-containingmolecule (and other secondary changes resulting from regulation of thatprotein). In other embodiments, the test and control cells may not besubstantially identical but, in these cases, the genetic differences(besides any exogenous ZFP-coding polynucleotides) are typically known.

[0116] Control cells and test cells used in the present methods can beindividual cells or cell populations, the latter being more usual. Thecell types can be cell lines or natural (e.g., isolated) cells such as,for example, primary cells. Cell lines are available, for example fromthe American Type Culture Collection (ATCC), or can be generated bymethods known in the art, as described for example in Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique, 3rd ed., 1994, andreferences cited therein. Similarly cells can be isolated by methodsknown in the art. Other non-limiting examples of cell types includecells that have or are subject to pathologies, such as cancerous cellsand transformed cells, pathogenically infected cells, stem cells, fullydifferentiated cells, partially differentiated cells, immortalized cellsand the like. Both prokaryotic (e.g., bacterial) and eukaryotic (e.g.,yeast, plant, fungal, piscine and mammalian cells such as feline,canine, murine, bovine, porcine and human) cells can be used, witheukaryotic cells being preferred. Mammalian (human and non-human) celltypes are particularly preferred. Archaeal cells can also be used. Thechoice of cell type depends in part on the intended recipient of thedrug being tested. For example, human cell types are advantageous forscreening drugs intended for use in human, and feline cell types areadvantageous for screening drugs intended for use in cats.

[0117] Suitable mammalian cell lines include CHO (chinese hamster ovary)cells, HEP-G2 cells, BaF-3 cells, Schneider cells, COS cells (monkeykidney cells expressing SV40 T-antigen), CV-1 cells, HuTu80 cells,NTERA2 cells, NB4 cells, HL-60 cells and HeLa cells, 293 cells (see,e.g., Graham et al. (1977) J. Gen. Virol. 36:59), and myeloma cells likeSP2 or NS0 (see, e.g., Galfre and Milstein (1981) Meth. Enzymol.73(B):3-46. Other eukaryotic cells include, for example, insect (e.g.,sp. frugiperda), fungal cells, including yeast (e.g., S. cerevisiae, S.pombe, P. pastoris, K. lactis, H. polymorpha), and plant cells (Fleer,R. (1992) Current Opinion in Biotechnology 3:486-496). Bacterial celltypes include E. coli, B. subtilis and S. typhimurium.

[0118] The cells can be transiently or stably transfected or transformedwith the ZFP or fusion molecule (or polynucleotide encoding the ZFP orfusion molecule). Methods of transfecting cells are known in the art anddescribed for example in Ausubel et al., supra. Example 11 shows how 293cells can be stably transfected with a nucleic acid molecule encoding aZFP that activates erythropoietin (EPO) expression.

[0119] Furthermore, as noted above, zinc finger proteins can be designedto suppress or activate expression of essentially any cellular gene.Accordingly, ZFPs and ZFP fusions can be designed to repress expressionof a protein that is structurally related to an intended moleculartarget or to overexpress a molecular target, to generate cells suitablefor cell-based HTS assays.

[0120] One can use a number of methods in applying zincfinger-containing cells to cell-based assays for drug discovery. Thetest cell population and the control cell population, for example, canbe put in two different vessels. A solution of a candidate compound canbe added sequentially to both vessels and a cellular property for eachcell population quantified and/or observed. When there is a significantdifference (i. e., outside the scope of experimental error) betweenvalues of the cellular property for the respective cell populations, onedetermines the candidate compound to be a “hit” in the assay.

[0121] Another method involves first contacting the control cellpopulation with a candidate compound and measuring a particular cellularresponse. The value of the response serves as a baseline measure for theassay. One then contacts the test cell population with the candidatecompound and measures the same cellular response. A statisticallydifferent value for the two responses indicates that the compound is a“hit”; it substantially interacts with the molecular target. This methodcan also be done in the opposite order, where one first assays the testcell population.

[0122] In other methods, zinc finger proteins are used to enhanceexpression of a molecular target in a test cell. Such test cells canoptionally be used in conjunction with control cells in which the targetis expressed at normal levels or other test cells in which the target isexpressed at subnormal levels. Alternatively, test cells in whichexpression of a molecular target is enhanced can be used withoutcomparison to control cells. For example, one can perform an analysis inwhich cellular response is measured in the presence and absence of acompound.

[0123] In some methods, analysis of cellular response in test andcontrol cells is performed in parallel. In other methods, analysis ofcellular response in test cells is compared with historical controls. Insome methods, cellular response in the presence of a compound in eithertest or control cells is compared with the response of like cells inabsence of the compound.

[0124] Virtually any component of a cell can serve as a molecular targetfor drug screening. Typically, the molecular target is a polypeptide.Non-limiting examples of molecular targets of interest include growthfactor receptors (e.g., FGFR, PDGFR, EFG, NGFR, and VEGF). Other targetsare G-protein receptors and include substance K receptor, theangiotensin receptor, the α- and β-adrenergic receptors, the serotoninreceptors, and PAF receptor. See, e.g., Gilman, Ann. Rev. Biochem.56:625-649 (1987). Other targets include ion channels (e.g., calcium,sodium, potassium channels), muscarinic receptors, acetylcholinereceptors, GABA receptors, glutamate receptors, and dopamine receptors(see Harpold, 5,401,629 and U.S. Pat. No. 5,436,128). Other targets areadhesion proteins such as integrins, selectins, and immunoglobulinsuperfamily members (see Springer, Nature 346:425-433 (1990). Osborn,Cell 62:3 (1990); Hynes, Cell 69:11 (1992)). Other targets arecytokines, such as interleukins IL-1 through IL-13, tumor necrosisfactors α & β, interferons α, β and γ, transforming growth factor Beta(TGF-β), colony stimulating factor (CSF) and granulocyte/macrophagecolony stimulating factor (GM-CSF). See Human Cytokines: Handbook forBasic & Clinical Research (Aggrawal et al. eds., Blackwell Scientific,Boston, Mass. 1991). Other targets are hormones, enzymes, andintracellular and intercellular messengers, such as, adenyl cyclase,guanyl cyclase, and phospholipase C, nuclear receptors (e.g., FXR(Farnesoid X Receptor), PPARs (Peroxisome Proliferator ActivatorReceptors), and RZR (Retinoid Z Receptor)), and organelle receptors.Target molecules can be human, mammalian viral, plant, fungal orbacterial. Other targets are antigens, such as proteins, glycoproteinsand carbohydrates from microbial pathogens, both viral and bacterial,and tumors. Still other targets are described in U.S. Pat. No.4,366,241. Some compound screened for interaction with a target merelybind to a target. Other compounds agonize or antagonize the target.

[0125] In some methods, compounds are screened individually. In othermethods, many compounds are screened in parallel. Microtiter plates androbotics are particularly useful for parallel screening of manycompounds. Optical detection is also preferred for rapidity andautomation. Hundreds, thousands or even millions of compounds can bescreened per week.

[0126] Once a “hit” is identified using the present methods derivativesof the compound can be made to maximize its ability to interact with themolecular target. Derivatives can be produced using conventionaltechniques such as self-consistent field (SCF) analysis, configurationinteraction (CI) analysis, and normal mode dynamics analysis. Computerprograms for implementing these techniques are readily available. SeeRein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions(Alan Liss, New York, 1989). Compound derivatives are subjected torescreening in the cell-based assay to select the one(s) thatdemonstrate the best interaction profile with the molecular target.

[0127] In other embodiments, the cells are engineered such that areporter system is operably linked to signal transduction through amolecular target. Reporter expression can be directly detected bydetecting formation of transcript or of translation product. Forexample, transcription product can be detected using RNA (Northern)blots and the formation of certain proteins can be detected using acharacteristic stain or by detecting an inherent characteristic of theprotein. More typically, however, expression of reporter is determinedby detecting a product formed as a consequence of an activity of thereporter.

[0128] Thus, cell based screening assays can be performed in which ZFPsmediate a positive response (e.g., activation of a reporter and/orselectable marker) to a negative signal, for example release of areporter or selectable marker from ZFP-controlled repression as a resultof blockage of an event in a signal transduction cascade. For instance,to screen a compound for its effect on a known signal transductioncascade, the compound is administered to a cell that is known to expressa target that is involved in a particular signal transduction cascade.In certain embodiments, the target is a cell surface or membrane-boundreceptor so that the compound does not need to traverse the cellmembrane or wall. In other embodiments, the target is an intracellularcomponent of the signal transduction cascade and, accordingly, thecompound can be also be administered directly into the cell usingmethods known in the art and described herein.

[0129] Typically, in these positive-response assays, the test cellcomprises (1) a polynucleotide encoding a fusion molecule comprising anexogenous zinc finger protein and a functional domain (e.g., arepression domain) and (2) a polynucleotide encoding a reporter and/orselectable molecule (e.g., green fluorescent protein, drug resistance,etc.). The polynucleotide encoding the fusion molecule is operablylinked to transcriptional control elements responsive to the signaltransduction cascade of interest. In embodiments in which the functionaldomain is a repression domain, the fusion molecule is designed suchthat, when expressed, it represses expression of the reporter molecule.Thus, when the signal transduction cascade is functioning, the fusionmolecule is expressed and, in turn, it serves to repress the expressionof the reporter molecule. If a test compound interferes with expressionof the repressive fusion molecule, for example by blocking a componentof the signal transduction pathway that acts, directly or indirectly, onthe control elements which regulate transcription of the fusionmolecule, repression of reporter molecule expression is diminished andincreased levels of reporter expression are observed. Thus, any compoundcan be screened for its ability to interfere with a signal transductioncascade by monitoring reporter levels or using positive and/or negativeselections to monitor modulation of the reporter (e.g., selectablemarker) molecule by the ZFP.

[0130] Any of the methods described herein can be used with any reporterand/or selectable marker. Reporters that can be directly detectedinclude fluorescent molecules such as, for example, GFP (greenfluorescent protein). Fluorescence is detected using a variety ofcommercially available fluorescent detection systems, including afluorescence-activated cell sorter (FACS) system for example. Otherreporters are enzymes that catalyze the formation of a detectableproduct. Suitable enzymes include proteases, nucleases, lipases,phosphatases, sugar hydrolases and esterases. Examples of suitablereporter genes that encode enzymes include, for example, CAT(chloramphenicol acetyl transferase; Alton and Vapnek (1979) Nature282:864-869), luciferase, β-galactosidase, β-glucuronidase, β-lactamase,horseradish peroxidase and alkaline phosphatase (e.g., Toh, et al.(1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol.Appl. Gen. 2:101).

[0131] Selectable markers form a subset or reporter molecules and canalso be used instead of, or in addition to, the directly detectablereporters described above. Positive selection markers are thosepolynucleotides that encode a product that enables only cells that carryand express the gene to survive and/or grow under certain conditions.For example, cells that express neomycin resistance (Neo^(r)) gene areresistant to the compound G418, while cells that do not express Neo^(r)are skilled by G418. Other examples of positive selection markersincluding hygromycin resistance, Zeocin® resistance and the like will beknown to those of skill in the art. Negative selection markers are thosepolynucleotides that encode a produce that enables only cells that carryand express the gene to be killed under certain conditions. For example,cells that express thymidine kinase (e.g., herpes simplex virusthymidine kinase, HSV-TK) are killed when gancyclovir is added. Othernegative selection markers are known to those skilled in the art. Theselectable marker need not be a transgene and, additionally, reportersand selectable markers can be used in various combinations.

[0132] D. Biochemical Assays

[0133] Also described herein are methods of performing biochemicaldrug-screening assays. An important biochemical assay to test for drugsthat affect a specific receptor involves isolation of cell membranescomprising the receptor of interest and testing for binding of candidatecompounds. Thus, cells that overexpress particular proteins associatedwith cell membranes (e.g., receptors) can be readily generated usingzinc finger proteins that activate expression of these proteins.Subsequently, the membranes of the cells that overexpress the protein ofinterest can be readily isolated for use in assays, for example bindingassays.

[0134] E. Compounds and Cell Properties

[0135] The methods described herein are useful in screening a widevariety of compounds. For example, compounds to be screened in thepresent methods can be from combinatorial libraries of peptides or smallmolecules, hormones, growth factors, and cytokines, can be naturallyoccurring molecules, or can be from existing repertoires of chemicalcompounds synthesized by the pharmaceutical industry. Combinatoriallibraries can be produced for many types of compound that can besynthesized in a step-by-step fashion. Such compounds include, forexample, polypeptides, beta-turn mimetics, polysaccharides, nucleicacids, phospholipids, hormones, prostaglandins, steroids, aromaticcompounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines and oligocarbamates. Large combinatoriallibraries of the compounds can be constructed by the encoded syntheticlibraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503and Scripps, WO 95/30642 (each of which is incorporated by reference forall purposes). Peptide libraries can also be generated by phage displaymethods. See, e.g., Devlin, WO 91/18980. Compounds to be screened canalso be obtained from the National Cancer Institute's Natural ProductRepository, Bethesda, Md. Existing compounds or drugs with knownefficacy can also be screened to evaluate side effects and/or additionalindications.

[0136] Furthermore, a variety of cellular and/or biochemical responses(also termed cell properties) can be measured and compared in themethods described herein. In some methods, the value of the cellproperty is measured as a function of cell growth, neovascularization,hormone release, pH changes, changes in intracellular second messengerssuch as GMP, binding to receptor and the like.

[0137] In other embodiments, the cellular response to administration ofa compound is normally quantified as a value of a cellular property. Theunits of the value depend on the property. For example, the units can beunits of absorbance, photon count, radioactive particle count or opticaldensity.

[0138] Delivery of Molecules

[0139] When the molecular target is intracellular, a compound thatinteracts with it must traverse the cell membrane. A compound contactedwith a cell can cross the cell membrane in a number of ways. If thecompound has suitable size and charge properties, it can be passivelytransported across the membrane. Other processes of membrane passageinclude active transport (e.g., receptor mediated transport),endocytosis and pinocytosis. Where a compound cannot be effectivelytransported by any of the preceding methods, microinjection, biolisticsor other methods can be used to deliver it to the internal portion ofthe cell. Alternatively, if the compound to be screened is a protein, anucleic acid encoding the protein can be introduced into the cell andexpressed within the cell.

[0140] Likewise, the exogenous zinc finger protein that effectsregulation within a cell must be introduced into the cell. Typicallysuch is achieved by introducing either the ZFP molecule or a nucleicacid encoding the ZFP into the cell resulting in expression of the zincfinger protein within the cell. Nucleic acids can be introduced byconventional means including viral based methods, chemical methods,lipofection and microinjection. The introduced nucleic acid canintegrate into the host chromosome, persist in episomal form or can havea transient existence in the cytoplasm. Similarly, an exogenous proteincan be introduced into a cell in protein form. For example, the zincfinger protein can be introduce by lipofection, biolistics, ormicroinjection or through fusion to membrane translocating domains.

[0141] Thus, the compositions described herein can be provided to thetarget cell in vitro or in vivo. In addition, the compositions can beprovided as polypeptides, polynucleotides or combination thereof.

[0142] A. Delivery of Polynucleotides

[0143] In certain embodiments, the compositions are provided as one ormore polynucleotides. Further, as noted above, a zinc fingerprotein-containing composition can be designed as a fusion between apolypeptide zinc finger and a functional domain, that is encoded by afusion nucleic acid. In both fusion and non-fusion cases, the nucleicacid can be cloned into intermediate vectors for transformation intoprokaryotic or eukaryotic cells for replication and/or expression.Intermediate vectors for storage or manipulation of the nucleic acid orproduction of protein can be prokaryotic vectors, (e.g., plasmids),shuttle vectors, insect vectors, or viral vectors for example. A nucleicacid encoding a zinc finger protein can also cloned into an expressionvector, for administration to a bacterial cells, fungal cells, protozoalcells, plant cells, or animal cells such as piscine cells or mammaliancells, preferably a human cell.

[0144] To obtain expression of a cloned nucleic acid, it is typicallysubcloned into an expression vector that contains a promoter to directtranscription. Suitable bacterial and eukaryotic promoters are wellknown in the art and described, e.g., in Sambrook et al., supra; Ausubelet al., supra; and Kriegler, Gene Transfer and Expression: A LaboratoryManual (1990). Bacterial expression systems are available in, e.g., E.coli, Bacillus sp., and Salmonella. Palva et al. (1983) Gene 22:229-235.Kits for such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available, for example, fromInvitrogen, Carlsbad, Calif. and Clontech, Palo Alto, Calif.

[0145] The promoter used to direct expression of the nucleic acid ofchoice depends on the particular application. For example, a strongconstitutive promoter is typically used for expression and purification.In contrast, when a protein is to be used in vivo, either a constitutiveor an inducible promoter is used, depending on the particular use of theprotein. In addition, a weak promoter can be used, such as HSV TK or apromoter having similar activity. The promoter typically can alsoinclude elements that are responsive to transactivation, e.g., hypoxiaresponse elements, Gal4 response elements, lac repressor responseelement, and small molecule control systems such as tet-regulatedsystems and the RU-486 system. See, e.g., Gossen et al. (1992) Proc.Natl. Acad. Sci USA 89:5547-5551; Oligino et al.(1998) Gene Ther.5:491-496; Wang et al. (1997) Gene Ther. 4:432-441; Neering et al.(1996) Blood 88:1147-1155; and Rendahl et al. (1998) Nat. Biotechnol.16:757-761.

[0146] In addition to a promoter, an expression vector typicallycontains a transcription unit or expression cassette that containsadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to the nucleicacid sequence, and signals required, e.g., for efficient polyadenylationof the transcript, transcriptional termination, ribosome binding, and/ortranslation termination. Additional elements of the cassette mayinclude, e.g., enhancers, and heterologous spliced intronic signals.

[0147] The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe resulting ZFP polypeptide, e.g., expression in plants, animals,bacteria, fungi, protozoa etc. Standard bacterial expression vectorsinclude plasmids such as pBR322, pBR322-based plasmids, pSKF, pET23D,and commercially available fusion expression systems such as GST andLacZ. Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, for monitoring expression, and formonitoring cellular and subcellular localization, e.g., c-myc or FLAG.

[0148] Regulatory elements from eukaryotic viruses are often used ineukaryotic expression vectors, e.g., SV40 vectors, papilloma virusvectors, and vectors derived from Epstein-Barr virus. Other exemplaryeukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV40 early promoter, SV40 late promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

[0149] Some expression systems have markers for selection of stablytransfected cell lines such as thymidine kinase, hygromycin Bphosphotransferase, and dihydrofolate reductase. High-yield expressionsystems are also suitable, such as baculovirus vectors in insect cells,with a nucleic acid sequence coding for a ZFP as described herein underthe transcriptional control of the polyhedrin promoter or any otherstrong baculovirus promoter.

[0150] Elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli (or in the prokaryotichost, if other than E. coli), a selective marker, e.g., a gene encodingantibiotic resistance, to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the vector to allow insertion of recombinant sequences.

[0151] Standard transfection methods can be used to produce bacterial,mammalian, yeast, insect, or other cell lines that express largequantities of zinc finger proteins, which can be purified, if desired,using standard techniques. See, e.g., Colley et al. (1989) J. Biol.Chem. 264:17619-17622; and Guide to Protein Purification, in Methods inEnzymology, vol. 182 (Deutscher, ed.) 1990. Transformation of eukaryoticand prokaryotic cells are performed according to standard techniques.See, e.g., Morrison (1977) J. Bacteriol. 132:349-351; Clark-Curtiss etal. (1983) in Methods in Enzymology 101:347-362 (Wu et al., eds).

[0152] Any procedure for introducing foreign nucleotide sequences intohost cells can be used. These include, but are not limited to, the useof calcium phosphate transfection, DEAE-dextran-mediated transfection,polybrene, protoplast fusion, electroporation, lipid-mediated delivery(e.g., liposomes), microinjection, particle bombardment, introduction ofnaked DNA, plasmid vectors, viral vectors (both episomal andintegrative) and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the protein of choice.

[0153] Conventional viral and non-viral based nucleic acid deliverymethods can be used to introduce nucleic acids into host cells or targettissues. Such methods can be used to administer nucleic acids to cellsin vitro. Additionally, nucleic acids are administered in vivo or exvivo. Non-viral vector delivery systems include DNA plasmids, nakednucleic acid, and nucleic acid complexed with a delivery vehicle such asa liposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For reviews of nucleic acid delivery procedures, see, for example,Anderson (1992) Science 256:808-813; Nabel et al. (1993) TrendsBiotechnol. 11:211-217; Mitani et al. (1993) Trends Biotechnol.11:162-166; Dillon (1993) Trends Biotechnol. 11:167-175; Miller (1992)Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154;Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer etal. (1995) British Medical Bulletin 51(1):31-44; Haddada et al., inCurrent Topics in Microbiology and Immunology, Doerfler and Böhm (eds),1995; and Yu et al. (1994) Gene Therapy 1:13-26.

[0154] Methods of non-viral delivery of nucleic acids includelipofection, microinjection, ballistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in, e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424 and WO 91/16024. Nucleic acid can bedelivered to cells (in vitro or ex vivo administration) or to targettissues (in vivo administration).

[0155] The preparation of lipid:nucleic acid complexes, includingtargeted liposomes such as immunolipid complexes, is well known to thoseof skill in the art. See, e.g., Crystal (1995) Science 270:404-410;Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994)Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem.5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992)Cancer Res. 52:4817-4820; and U.S. Pat. Nos. 4,186,183; 4,217,344;4,235,871; 4,261,975; 4,485,054; 4,501,728; 4,774,085; 4,837,028 and4,946,787.

[0156] The use of RNA or DNA virus-based systems for the delivery ofnucleic acids take advantage of highly evolved processes for targeting avirus to specific cells in the body and trafficking the viral payload tothe nucleus. Viral vectors can be administered directly to subjects (invivo) or they can be used to treat cells in vitro or ex vivo.Conventional viral based systems for the delivery of ZFPs includeretroviral, lentiviral, poxviral, adenoviral, adeno-associated viral,vesicular stomatitis viral and herpesviral vectors. Integration in thehost genome is possible with certain viral vectors, including theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

[0157] The tropism of a retrovirus can be altered by incorporatingforeign envelope proteins, allowing alteration and/or expansion of thepotential target cell population. Lentiviral vectors are retroviralvector that are able to transduce or infect non-dividing cells andtypically produce high viral titers. Selection of a retroviral nucleicacid delivery system would therefore depend on the target cell and/ortissue. Retroviral vectors have a packaging capacity of up to 6-10 kb offoreign sequence and are comprised of cis-acting long terminal repeats(LTRs). The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate the exogenousgene into the target cell to provide permanent transgene expression.Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), and combinationsthereof. Buchscher et al. (1992) J. Virol. 66:2731-2739; Johann et al.(1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol.176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al.(1991) J. Virol. 65:2220-2224; and PCT/US94/05700). pLASN and MFG-S areexamples of retroviral vectors that have been used in clinical trials.Dunbar et al. (1995) Blood 85:3048-305; Kohn et al. (1995) Nature Med.1:1017-102; Malech et al. (1997) Proc. Natl. Acad. Sci. USA94:12133-12138. PA317/pLASN was the first therapeutic vector used in agene therapy trial. (Blaese et al. (1995) Science 270:475-480.Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. Ellem et al. (1997) Immunol Immunother. 44(1):10-20;Dranoff et al. (1997) Hum. Gene Ther. 1:111-2.

[0158] Adeno-associated virus (AAV) vectors are also used to transducecells with target nucleic acids, e.g., in the in vitro production ofnucleic acids and peptides, and for in vivo and ex vivo applications.See, e.g., West et al. (1987) Virology 160:38-47; U.S. Pat.No.4,797,368; WO 93/24641; Kotin (1994) Hum. Gene Ther. 5:793-801; andMuzyczka (1994) J. Clin. Invest. 94:1351. Construction of recombinantAAV vectors are described in a number of publications, including U.S.Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5:3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol. 4:2072-2081;Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; andSamulski et al. (1989) J. Virol. 63:3822-3828.

[0159] Recombinant adeno-associated virus vectors based on the defectiveand nonpathogenic parvovirus adeno-associated virus type 2 (AAV-2) are apromising nucleic acid delivery system. Exemplary AAV vectors arederived from a plasmid containing the AAV 145 bp inverted terminalrepeats flanking a transgene expression cassette. Efficient transfer ofnucleic acids and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.Wagner et al. (1998) Lancet 351 (9117):1702-3; and Kearns et al. (1996)Gene Ther. 9:748-755.

[0160] In applications for which transient expression is preferred,adenoviral-based systems are useful. Adenoviral based vectors arecapable of very high transduction efficiency in many cell types and arecapable of infecting, and hence delivering nucleic acid to, bothdividing and non-dividing cells. With such vectors, high titers andlevels of expression have been obtained. Adenovirus vectors can beproduced in large quantities in a relatively simple system.

[0161] Replication-deficient recombinant adenovirus (Ad) vectors can beproduced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; the replication defectorvector is propagated in human 293 cells that supply the required E1functions in trans. Ad vectors can transduce multiple types of tissuesin vivo, including non-dividing, differentiated cells such as thosefound in the liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity for inserted DNA. An example of the use of an Advector in a clinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection. Sterman et al. (1998) Hum.Gene Ther. 7:1083-1089. Additional examples of the use of adenovirusvectors for nucleic acid delivery include Rosenecker et al. (1996)Infection 24:5-10; Sterman et al., supra; Welsh et al. (1995) Hum. GeneTher. 2:205-218; Alvarez et al. (1997) Hum. Gene Ther. 5:597-613; andTopf et al. (1998) Gene Ther. 5:507-513.

[0162] Packaging cells are used to form virus particles that are capableof infecting a host cell. Such cells include 293 cells, which packageadenovirus, and Ψ2 cells or PA317 cells, which package retroviruses.Viral vectors used in nucleic acid delivery are usually generated by aproducer cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for the proteinto be expressed. Missing viral functions are supplied in trans, ifnecessary, by the packaging cell line. For example, AAV vectors used innucleic acid delivery typically only possess ITR sequences from the AAVgenome, which are required for packaging and integration into the hostgenome. Viral DNA is packaged in a cell line, which contains a helperplasmid encoding the other AAV genes, namely rep and cap, but lackingITR sequences. The cell line is also infected with adenovirus as ahelper. The helper virus promotes replication of the AAV vector andexpression of AAV genes from the helper plasmid. The helper plasmid isnot packaged in significant amounts due to a lack of ITR sequences.Contamination with adenovirus can be reduced by, e.g., heat treatment,which preferentially inactivates adenoviruses.

[0163] In many nucleic acid delivery applications, it is desirable thatthe vector be delivered with a high degree of specificity to aparticular tissue type. A viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al. (1995) Proc. Natl.Acad. Sci. USA 92:9747-9751 reported that Moloney murine leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., F_(ab) or F_(v)) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to non-viral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

[0164] Vectors can be delivered in vivo by administration to a subject,typically by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ortopical application, as described infra. Alternatively, vectors can bedelivered to cells in vitro or ex vivo, such as cells explanted from asubject (e.g., lymphocytes, bone marrow aspirates, tissue biopsy).

[0165] In one embodiment, hematopoietic stem cells are used in in vitroprocedures for cell transfection and nucleic acid delivery. Theadvantage to using stem cells is that they can be differentiated intoother cell types in vitro. Methods for differentiating CD34+ stem cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known. Inaba et al. (1992) J. Exp.Med. 176:1693-1702.

[0166] Stem cells are isolated for transduction and differentiationusing known methods. For example, stem cells are isolated from bonemarrow cells by panning the bone marrow cells with antibodies which bindunwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells),GR-1 (granulocytes), and Iad (differentiated antigen presenting cells).See Inaba et al., supra.

[0167] B. Delivery of Polypeptides

[0168] In other embodiments, ZFPs or ZFP fusion proteins areadministered directly to target cells. In certain in vitro situations,the target cells are cultured in a medium containing one or more fusionproteins comprising functional domains fused to one or more of the ZFPs.

[0169] An important factor in the administration of polypeptidecompounds is ensuring that the polypeptide has the ability to traversethe plasma membrane of a cell, or the membrane of an intra-cellularcompartment such as the nucleus. Cellular membranes are composed oflipid-protein bilayers that are freely permeable to small, nonioniclipophilic compounds and are inherently impermeable to polar compounds,macromolecules, and therapeutic or diagnostic agents. However, proteins,lipids and other compounds, which have the ability to translocatepolypeptides across a cell membrane, have been described.

[0170] For example, “membrane translocation polypeptides” haveamphiphilic or hydrophobic amino acid subsequences that have the abilityto act as membrane-translocating carriers. In one embodiment,homeodomain proteins have the ability to translocate across cellmembranes. The shortest internalizable peptide of a homeodomain protein,Antennapedia, was found to be the third helix of the protein, from aminoacid position 43 to 58. Prochiantz (1996) Curr. Opin. Neurobiol.6:629-634. Another subsequence, the h (hydrophobic) domain of signalpeptides, was found to have similar cell membrane translocationcharacteristics. Lin et al. (1995) J. Biol. Chem. 270:14255-14258.

[0171] Examples of peptide sequences which can be linked to a zincfinger polypeptide (or fusion containing the same) for facilitating itsuptake into cells include, but are not limited to: an 11 amino acidpeptide of the tat protein of HIV; a 20 residue peptide sequence whichcorresponds to amino acids 84-103 of the p16 protein (see Fahraeus etal. (1996) Curr. Biol. 6:84); the third helix of the 60-amino acid longhomeodomain of Antennapedia (Derossi et al. (1994) J. Biol. Chem.269:10444); the h region of a signal peptide, such as the Kaposifibroblast growth factor (K-FGF) h region (Lin et al., supra); and theVP22 translocation domain from HSV (Elliot et al. (1997) Cell88:223-233). Other suitable chemical moieties that provide enhancedcellular uptake can also be linked, either covalently or non-covalently,to the ZFPs.

[0172] Toxin molecules also have the ability to transport polypeptidesacross cell membranes. Often, such molecules (called “binary toxins”)are composed of at least two parts: a translocation or binding domainand a separate toxin domain. Typically, the translocation domain, whichcan optionally be a polypeptide, binds to a cellular receptor,facilitating transport of the toxin into the cell. Several bacterialtoxins, including Clostridium perfringens iota toxin, diphtheria toxin(DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillusanthracis toxin, and pertussis adenylate cyclase (CYA), have been usedto deliver peptides to the cell cytosol as internal or amino-terminalfusions. Arora et al. (1993) J. Biol. Chem. 268:3334-3341; Perelle etal. (1993) Infect. Immun. 61:5147-5156; Stenmark et al. (1991) J. CellBiol. 113:1025-1032; Donnelly et al. (1993) Proc. Natl. Acad. Sci. USA90:3530-3534; Carbonetti et al. (1995) Abstr. Annu. Meet. Am. Soc.Microbiol. 95:295; Sebo et al. (1995) Infect. Immun. 63:3851-3857;Klimpel et al. (1992) Proc. Natl. Acad. Sci. USA. 89:10277-10281; andNovak et al (1992) J. Biol. Chem. 267:17186-17193.

[0173] Such subsequences can be used to translocate polypeptides,including the polypeptides as disclosed herein, across a cell membrane.This is accomplished, for example, by derivatizing the fusionpolypeptide with one of these translocation sequences, or by forming anadditional fusion of the translocation sequence with the fusionpolypeptide. Optionally, a linker can be used to link the fusionpolypeptide and the translocation sequence. Any suitable linker can beused, e.g., a peptide linker.

[0174] A suitable polypeptide can also be introduced into an animalcell, preferably a mammalian cell, via liposomes and liposomederivatives such as immunoliposomes. The term “liposome” refers tovesicles comprised of one or more concentrically ordered lipid bilayers,which encapsulate an aqueous phase. The aqueous phase typically containsthe molecule(s) to be delivered to the cell.

[0175] The liposome fuses with the plasma membrane, thereby releasingthe molecule(s) into the cytosol. Alternatively, the liposome isphagocytosed or taken up by the cell in a transport vesicle. Once in theendosome or phagosome, the liposome is either degraded or it fuses withthe membrane of the transport vesicle and releases its contents.

[0176] In current methods of drug delivery via liposomes, the liposomeultimately becomes permeable and releases the encapsulated molecule(s)at the target tissue or cell. For systemic or tissue specific delivery,this can be accomplished, for example, in a passive manner wherein theliposome bilayer is degraded over time through the action of variousagents in the body. Alternatively, active drug release involves using anagent to induce a permeability change in the liposome vesicle. Liposomemembranes can be constructed so that they become destabilized when theenvironment becomes acidic near the liposome membrane. See, e.g., Proc.Natl. Acad. Sci. USA 84:7851(1987); Biochemistry 28:908 (1989). Whenliposomes are endocytosed by a target cell, for example, they becomedestabilized and release their contents. This destabilization is termedfusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basis ofmany “fusogenic” systems.

[0177] For use with the methods and compositions disclosed herein,liposomes typically comprise a fusion polypeptide as disclosed herein, alipid component, e.g., a neutral and/or cationic lipid, and optionallyinclude a receptor-recognition molecule such as an antibody that bindsto a predetermined cell surface receptor or ligand (e.g., an antigen). Avariety of methods are available for preparing liposomes as describedin, e.g.; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975;4,485,054; 4,501,728; 4,774,085; 4,837,028; 4,235,871; 4,261,975;4,485,054; 4,501,728; 4,774,085; 4,837,028; 4,946,787; PCT PublicationNo. WO 91/17424; Szoka et al. (1980) Ann. Rev. Biophys. Bioeng. 9:467;Deamer et al. (1976) Biochim. Biophys. Acta 443:629-634; Fraley, et al.(1979) Proc. Natl. Acad. Sci. USA 76:3348-3352; Hope et al. (1985)Biochim. Biophys. Acta 812:55-65; Mayer et al. (1986) Biochim. Biophys.Acta 858:161-168; Williams et al. (1988) Proc. Natl. Acad. Sci. USA85:242-246; Liposomes, Ostro (ed.), 1983, Chapter 1); Hope et al. (1986)Chem. Phys. Lip. 40:89; Gregoriadis, Liposome Technology (1984) andLasic, Liposomes: from Physics to Applications (1993). Suitable methodsinclude, for example, sonication, extrusion, highpressure/homogenization, microfluidization, detergent dialysis,calcium-induced fusion of small liposome vesicles and ether-fusionmethods, all of which are well known in the art.

[0178] In certain embodiments, it may be desirable to target a liposomeusing targeting moieties that are specific to a particular cell type,tissue, and the like. Targeting of liposomes using a variety oftargeting moieties (e.g., ligands, receptors, and monoclonal antibodies)has been previously described. See, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044.

[0179] Examples of targeting moieties include monoclonal antibodiesspecific to antigens associated with neoplasms, such as prostate cancerspecific antigen and MAGE. Tumor cells can also be targeted by detectinggene products resulting from the activation or over-expression ofoncogenes, such as ras or c-erbB2. In addition, many tumors expressantigens normally expressed by fetal tissue, such as thealphafetoprotein (AFP) and carcinoembryonic antigen (CEA). Virallyinfected cells can be targeted using various viral antigens such ashepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens,Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV-1)and papilloma virus antigens. Inflammed cells can be targeted usingmolecules specifically recognized by surface molecules which areexpressed at sites of inflammation such as integrins (e.g., VCAM-1),selectin receptors (e.g., ELAM-1) and the like.

[0180] Standard methods for coupling targeting agents to liposomes areused. These methods generally involve the incorporation into liposomesof lipid components, e.g., phosphatidylethanolamine, which can beactivated for attachment of targeting agents, or incorporation ofderivatized lipophilic compounds, such as lipid derivatized bleomycin.Antibody targeted liposomes can be constructed using, for instance,liposomes which incorporate protein A. See Renneisen et al. (1990) J.Biol. Chem. 265:16337-16342 and Leonetti et al. (1990) Proc. Natl. Acad.Sci. USA 87:2448-2451.

[0181] Kits

[0182] Also provided are kits for performing any of the above methods.The kits typically contain cells for use in the above methods orcomponents for making such cells. For example, some kits contain pairsof test and control cells differing in that one cell population istransformed with an exogenous nucleic acid encoding a zinc fingerprotein designed to regulate expression of a molecular target (e.g., aprotein) within the test cells. Some kits contain a single cell type andother components that allow one to produce test cells from that celltype. Such components can include a vector encoding a zinc fingerprotein or the zinc finger protein itself. The kits can also containnecessary containers, buffers for transformation of cells, culture mediafor cells, and/or buffers for performing assays. Typically, the kitsalso contain a label indicating that the cells are to be used forscreening compounds. A label includes any material such as instructions,packaging or advertising leaflet that is attached to or otherwiseaccompanies the other components of the kit.

EXAMPLES

[0183] The following examples are offered to illustrate, but are not inany way intended to be limiting.

Example 1

[0184] This first Example demonstrates the construction of ZFPs designedto recognize DNA sequences contained in the promoter of the humanvascular endothelial growth factor (VEGF) gene. VEGF is an approximately46 kDa glycoprotein that is an endothelial cell-specific mitogen inducedby hypoxia. VEGF has been implicated in angiogenesis associated withcancer, various retinopathies, and other serious diseases. The DNAtarget site chosen was a region surrounding the transcription initiationsite of the gene. The two 9 base pair (bp) sites chosen are found withinthe sequence agcGGGGAGGATcGCGGAGGCTtgg (SEQ ID NO: 1), where theupper-case letters represent actual 9-bp targets. The protein targetingthe upstream 9-bp target was denoted VEGF1, and the protein targetingthe downstream 9-bp target was denoted VEGF3a. The major start site oftranscription for VEGF is at the T at the 3′ end of the first 9-bptarget, which is underlined in the sequence above.

[0185] The human SP-1 transcription factor was used as a progenitormolecule for the construction of designed ZFPs. SP-1 has a three fingerDNA-binding domain related to the well-studied murine Zif268 (Christy etal., PNAS 85:7857-7861 (1988)). Site-directed mutagenesis experimentsusing this domain have shown that the proposed “recognition rules” thatoperate in Zif268 can be used to adapt SP-1 to other target DNAsequences (Desjarlais & Berg, PNAS 91:11099-11103 (1994)). The SP-1sequence used for construction of zinc finger clones corresponds toamino acids 533 to 624 in the SP-1 transcription factor.

[0186] The selection of amino acids in the recognition helices of thetwo designed ZFPs, VEGF1 and VEGF3a, is summarized in Table 1. TABLE 1Amino acids chosen for recognition helices of VEGF-recognizing ZFPsPosition: Finger 1 Finger 2 Finger 3 Protein −1 2 3 6 −1 2 3 6 −1 2 3 6VEGF1 T S N R R S N R R D H R VEGF3A Q S D R R S N R R D E R

[0187] Coding sequences were constructed to express these peptides usinga PCR-based assembly procedure that utilizes six overlappingoligonucleotides. Three oligonucleotides corresponding to “universal”sequences that encode portions of the DNA-binding domain between therecognition helices. These oligonucleotides remain constant for any zincfinger construct. Three “specific” oligonucleotides were designed toencode the recognition helices. These oligonucleotides containedsubstitutions at positions −1, 2, 3 and 6 on the recognition helices tomake them specific for each of the different DNA-binding domains. Codonbias was chosen to allow expression in both mammalian cells and E. coli.

[0188] The PCR synthesis was carried out in two steps. First, the doublestranded DNA template was created by combining the six oligonucleotides(three universal, three specific) and using a four cycle PCR reactionwith a low temperature (25° C.) annealing step. At this temperature, thesix oligonucleotides join to form a DNA “scaffold.” The gaps in thescaffold were filled in by a combination of Taq and Pfu polymerases. Inthe second phase of construction, the zinc finger template was amplifiedin thirty cycles by external primers that were designed to incorporaterestriction sites for cloning into pUC19. Accuracy of clones for theVEGF ZFPs were verified by DNA sequencing. The DNA sequences of each ofthe two constructs are listed below. VEGF1:GGTACCCATACCTGGCAAGAAGAAGCAGCACATCTGCCACATCCAGGGCTGT (SEQ ID NO:2)GGTAAAGTTTACGGCACAACCTCAAATCTGCGTCGTCACCTGCGCTGGCACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACGCTTCACCCGTTCGTCAAACCTGCAGCGTCACAAGCGTACCCACACCGGTGAGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGTAGTGACCACCTGTCCCGTCACATCAAGACCCACCAGAATAAGAAGGGTGGATCC

[0189] VEGF1 translation:VPIPGKKKQHICHIQGCGKVYGTTSNLRRHLRWHTGERPFMCTWSYCGKRFTRS (SEQ ID NO:3)SNLQRHKRTHTGEKKFACPECPKRFMRSDHLSRHIKTHQNKKGGS

[0190] VEGF3a: GGTACCCATACCTGGCAAGAAGAAGCAGCACATCTGCCACATCCAGGGCTGT (SEQID NO:4) GGTAAAGTTTACGGCCAGTCCTCCGACCTGCAGCGTCACCTGCGCTGGCACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACGCTTCACCCGTTCGTCAAACCTACAGAGGCACAAGCGTACACACACCGGTGAGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGAAGTGACGAGCTGTCACGACATATCAAGACCCACCAGAACAAGAAGGGTGGATCC

[0191] VEGF3a translation:VPIPGKKKQHICHIQGCGKVYGQSSDLQRHLRWHTGERPFMCTWSYCGKRFTRS (SEQ ID NO:5)SNLQRHKRTHTGEKKFACPECPKRFMRSDELSRHIKTHQNKKGGS

[0192] The ability of the designed ZFPs to bind their target sites wasverified by expressing and purifying recombinant protein from E. coliand performing electrophoretic mobility shift assays (EMSAs). Theexpression of ZFPs was carried out in two different systems. In thefirst, the DNA-binding peptides were expressed in E. coli by insertingthem into the commercially available pET15b vector (Novagen). Thisvector contains a T7 promoter sequence to drive expression of therecombinant protein. The constructs were introduced into E. coliBL21/DE3 (lacI^(q)) cells, which contain an IPTG-inducible T7polymerase. Cultures were supplemented with 50 μM ZnCl₂, were grown at37° C. to an OD at 600 nm of 0.5-0.6, and protein production was inducedwith IPTG for 2 hrs. ZFP expression was seen at very high levels,approximately 30% of total cellular protein. These proteins are referredto as “unfused” ZFPs.

[0193] Partially pure unfused ZFPs were produced as follows (adaptedfrom Desjarlais & Berg, Proteins: Structure, Function and Genetics12:101-104 (1992)). A frozen cell pellet was resuspended in {fraction(1/50)}th volume of 1 M NaCl, 25 mM Tris HCl (pH 8.0), 100 μM ZnCl₂, 5mM DTT. The samples were boiled for 10 min. and centrifuged for 10 min.at ˜3,000×g. At this point the ZFP protein in the supernatant was>50%pure as estimated by staining of SDS polyacrylamide gels with Coomassieblue, and the product migrated at the predicted molecular weight ofaround 11 kDa.

[0194] The second method of producing ZFPs was to express them asfusions to the E. coli Maltose Binding Protein (MBP). N-terminal MBPfusions to the ZFPs were constructed by PCR amplification of the pET15bclones and insertion into the vector pMal-c2 under the control of theTac promoter (New England Biolabs). The fusion allows simplepurification and detection of the recombinant protein. It had beenreported previously that zinc finger DNA-binding proteins can beexpressed from this vector in soluble form to high levels in E. coli andcan bind efficiently to the appropriate DNA target without refolding(Liu et al. PNAS 94:5525-5530 (1997)). Production of MBP-fused proteinswas as described by the manufacturer (New England Biolabs).Transformants were grown in LB medium supplemented with glucose andampicillin, and were induced with IPTG for 3 hrs at 37° C. The cellswere lysed by French press, then exposed to an agarose-based amyloseresin, which specifically binds to the MBP moiety, thus acting as anaffinity resin for this protein. The MBP fusion protein was eluted with10 mM maltose to release ZFP of>50% purity. In some cases, the proteinswere further concentrated using a Centricon 30 filter unit (Amicon).

[0195] Partially purified unfused and MBP fusion ZFPs were tested byEMSA to assess binding to their target DNA sequences. The proteinconcentrations in the preparations were measured by Bradford assay(BioRad). Since SDS polyacrylamide gels demonstrated>50% homogeneity byeither purification method, no adjustment was made for ZFP purity in thecalculations. In addition, there could be significant amounts ofinactive protein in the preparations. Therefore, the data generated byEMSAs below represent an underestimate of the true affinity of theproteins for their targets (i.e., overestimate of K_(d)s). Two separatepreparations were made for each protein to help control for differencesin ZFP activity.

[0196] The VEGF DNA target sites for the EMSA experiments were generatedby embedding the 9-bp binding sites in 29-bp duplex oligonucleotides.The sequences of the recognition (“top”) strand and their complements(“bottom”) used in the assays are as follows:

[0197] VEGF site 1, top: 5′-CATGCATAGCGGGGAGGATCGCCATCGAT (SEQ ID NO:6)

[0198] VEGF site 1, bottom: 5′-ATCGATGGCGATCCTCCCCGCTATGCATG (SEQ IDNO:7)

[0199] VEGF site 3, top: 5′-CATGCATATCGCGGAGGCTTGGCATCGAT (SEQ ID NO:8)

[0200] VEGF site 3, bottom: 5′-ATCGATGCCAAGCCTCCGCGATATGCATG (SEQ IDNO:9)

[0201] The VEGF DNA target sites are underlined. The 3 bp on either sideof the 9 bp binding site was also derived from the actual VEGF DNAsequence. The top strand of each target site was labeled withpolynucleotide kinase and γ-³²P ATP. Top and bottom strands wereannealed in a reaction containing each oligonucleotide at 0.5 μM, 10 mMTris-HCl (pH 8.0), 1 mM EDTA, and 50 mM NaCl. The mix was heated to 95°C. for 5 min. and slow cooled to 30° C. over 60 min. Duplex formationwas confirmed by polyacrylamide gel electrophoresis. Free label andssDNA remaining in the target preparations did not appear to interferewith the binding reactions.

[0202] Binding of the ZFPs to target oligonucleotides was performed bytitrating protein against a fixed amount of duplex substrate. Twentymicroliter binding reactions contained 10 fmole (0.5 nM) 5′ ³²P-labeleddouble-stranded target DNA, 35 mM Tris HCl (pH 7.8), 100 mM KCl, 1 mMMgCl₂, 1 mM dithiothreitol, 10% glycerol, 20 μg/ml poly dI-dC(optionally), 200 μg/ml bovine serum albumin, and 25 μM ZnCl₂. Proteinwas added as one fifth volume from a dilution series made in 200 mMNaCl, 20 mM Tris (pH 7.5), 1 mM DTT. Binding was allowed to proceed for30 min. at room temperature. Polyacrylamide gel electrophoresis wascarried out at 4° C. using precast 10% or 10-20% Tris-HCl gels (BioRad)and standard Tris-Glycine running buffer containing 0.1 mM ZnCl₂.

[0203] A typical EMSA using an MBP fused ZFP was performed. In thiscase, a 3-fold dilution series of the MBP-VEGF1 protein was used. Theshifted product was quantitated on a phosphorimager (Molecular Dynamics)and the relative signal (percent of plateau value) vs. the log10 of nMprotein concentration was plotted. An apparent K_(d) was found bydetermining the protein concentration that gave half maximal binding ofMBP-VEGF1 to its target site, which in this experiment was approximately2 nM.

[0204] The binding affinities determined for the VEGF proteins can besummarized as follows. VEGF1 showed the stronger DNA-binding affinity;in multiple EMSA analyses, the average apparent K_(d) was determined tobe approximately 10 nM when bound to VEGF site 1. VEGF3a bound well toits target site but with a higher apparent K_(d) than VEGF1; the averageK_(d) for VEGF3a was about 200 nM. In both cases the MBP-fused andunfused versions of the proteins bound with similar affinities. K_(d)swere also determined under these conditions for MBP fusions of thewild-type Zif268 and SP-1 ZFPs, which yielded K_(d)s of 60 and 65 nM,respectively. These results are similar to binding constants reported inthe literature for Zif268 of approximately 2-30 nM (see, e.g., Jamiesonet al., Biochemistry 33:5689-5695 (1994)). The K_(d)s for the syntheticVEGF ZFPs therefore compare very favorably with those determined forthese naturally-occurring DNA-binding proteins.

[0205] In summary, this Example demonstrates the generation of two novelDNA-binding proteins directed to specific targets near thetranscriptional start of the VEGF gene. These proteins bind withaffinities similar to those of naturally-occurring transcription factorsbinding to their targets.

Example 2

[0206] If a zinc finger domain that recognizes a 9-bp target site lacksthe necessary affinity or specificity when expressed inside cells, alarger domain can be constructed to recognize an 18 base-pair targetsite, by joining separate three-finger domains with a linker sequence toform a six-finger protein. This should ensure that a functional domain(e.g., activator or repressor) fused to the ZFP is specifically targetedto the appropriate sequence, particularly under conditions in which onlysmall amounts of the ZFP fusion protein are being produced. The 9-bptarget sites in VEGF were chosen to be adjacent to one another so thatthe zinc fingers could be linked to recognize an 18-bp sequence. Thelinker DGGGS was chosen because it permits binding of ZFPs to two 9-bpsites that are separated by a one nucleotide gap, as is the case for theVEGF1 and VEGF3a sites (see also Liu et al., PNAS 5525-5530 (1997)).

[0207] The 6-finger VEGF3a/1 protein encoding sequence was generated asfollows. VEGF3a was PCR amplified using the primers SPE7(5′-GAGCAGAATTCGGCAAGAAGAAGCAGCAC, SEQ ID NO:10) and SPEamp12(5′-GTGGTCTAGACAGCTCGTCACTTCGC, SEQ ID NO:11) to generate EcoRI and XbaIrestriction sites at the ends (restriction sites underlined). VEGF1 wasPCR amplified using the primers SPEamp13(5′-GGAGCCAAGGCTGTGGTAAAGTTTACGG. SEQ ID NO:12) and SPEamp11(5′-GGAGAAGCTTGGATCCTCATTATCCC. SEQ ID NO:13) to generate StyI andHindIII restriction sites at the ends (restriction sites underlined).Using synthetic oligonucleotides, the following sequence was ligatedbetween the XbaI and StyI sites, where XbaI and StyI are underlined: TCTAGA CAC ATC AAA ACC CAC CAG AAC AAG AAA GAC GGC GGT GGC AGC GGC AAA AAGAAA CAG CAC ATA TGT CAC ATC CAA GG (SEQ ID NO:14). This introduced thelinker sequence DGGGS between the two SP-1 domains. The ligation productwas reamplified with primers SPE7 and SPEamp11 and cloned into pUC19using the EcoRI and HindIII sites. The linked ZFP sequences were thenamplified with primers

[0208] (1) GB19 GCCATGCCGGTACCCATACCTGGCAAGAAGAAGCAGCAC (SEQ ID NO:15)

[0209] (2) GB10 CAGATCGGATCCACCCTTCTTATTCTGGTGGGT (SEQ ID NO:16) tointroduce KpnI and BamHI sites for cloning into the modified pMAL-c2expression vector as described above.

[0210] The nucleotide sequence of the designed, 6-finger ZFP VEGF3a/1from KpnI to BamHI is:         GGTACCCATACCTGGCAAGAAGAAGCAGCACATCTGCCACATC (SEQ ID NO:17)CAGGGCTGTGGTAAAGTTTACGGCCAGTCCTCCGACCTGCAGCGTCACCTGCGCTGGCACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACGCTTCACACGTTCGTCAAACCTACAGAGGCACAAGCGTACACACACAGGTGAGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGAAGTGACGAGCTGTCTAGACACATCAAAACCCACCAGAACAAGAAAGACGGCGGTGGCAGCGGCAAAAAGAAACAGCACATATGTCACATCCAAGGCTGTGGTAAAGTTTACGGCACAACCTCAAATCTGCGTCGTCACCTGCGCTGGCACACCGGCGAGAGGCCTTTCATGTGTACCTGGTCCTACTGTGGTAAACGCTTCACCCGTTCGTCAAACCTGCAGCGTCACAAGCGTACCCACACCGGTGAGAAGAAATTTGCTTGCCCGGAGTGTCCGAAGCGCTTCATGCGTAGTGACCACCTGTCCCGTCACATCAAGACCCACCAGAATAAGAAGGGTGGATCC

[0211] The VEGF3a/1 amino acid translation (using single letter code)is:         VPIPGKKKQHICHIQGCGKVYGQSSDLQRHLRWHTGERIPFMCTWS (SEQ IDNO:18) YCGKRFTRSSNLQRHKRTHTGEKKFACPECPKRFMRSDELSRHIKTHQNKKDGGGSGKKKQHICHIQGCGKVYGTTSNLRRHLRWHTGERPFMCTWSYCGKRFTRSSNLQRHKRTHTGEKKFACPECPKRFMRSDHLSRHIKTHQNKKGGS

[0212] The 18-bp binding protein VEGF3a/1 was expressed in E. coli as anMBP fusion, purified by affinity chromatography, and tested in EMSAexperiments as described in Example 1. The target oligonucleotides wereprepared as described and comprised the following complementarysequences:

[0213] (1) JVF9 AGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAG (SEQ ID NO:19), and

[0214] (2) JVF10 CGCTCTACCCGGCTGCCCCAAGCCTCCGCGATCCTCCCCGCT (SEQ IDNO:20)

[0215] For the EMSA studies, 20 μl binding reactions contained 10 fmole(0.5 nM) 5′ ³²P-labeled double-stranded target DNA, 35 mM Tris HCl (pH7.8), 100 mM KCl, 1 mM MgCl₂, 5 mM dithiothreitol, 10% glycerol, 20μg/ml poly dI-dC, 200 μg/ml bovine serum albumin, and 25 μM ZnCl₂.Protein was added as one fifth volume from a 3-fold dilution series.Binding was allowed to proceed for 60 min at either room temperature or37° C. Polyacrylamide gel electrophoresis was carried out at roomtemperature or 37° C. using precast 10% or 10-20% Tris-HCl gels (BioRad)and standard Tris-Glycine running buffer. The room temperature assaysyielded an apparent K_(d) for this VEGF3a/1 protein of approximately 1.5nM. Thus, the 18-bp binding ZFP bound with high affinity to its targetsite. In a parallel experiment, VEGF1 protein was tested against itstarget using the oligonucleotides described in Example 1, yielding anapparent K_(d) of approximately 2.5 nM. When binding and electrophoresiswere performed at 37° C., the apparent K_(d) of VEGF3a/1 wasapproximately 9 nM when tested against the 18-bp target, compared to aK_(d) of 40 nM for VEGF1 tested against its target. This indicates thatthe difference in binding affinities is accentuated at the highertemperature.

[0216] The apparent K_(d) is a useful measure of the affinity of aprotein for its DNA target. However, for a DNA binding site either invitro or in vivo, its occupancy is determined to a large extent by theoff-rate of the DNA-binding protein. This parameter can be measured bycompetition experiments. The conditions for EMSA were as describedabove; binding and electrophoresis were performed at 37° C. These dataindicate that the half-life of the protein-DNA complex is more than tentimes longer for VEGF3a/1 than for VEGF1. Thus, under these in vitroconditions, the occupancy of the target site is much higher for the18-bp binding protein than for the 9-bp binding protein.

Example 3

[0217] This Example describes the construction of expression vectors forproducing ZFPs within mammalian cells, translocating them to thenucleus, and providing functional domains that are localized to thetarget DNA sequence by the ZFP. The functional domains employed are theKruppel-Associated Box (KRAB) repression domain and the Herpes SimplexVirus (HSV-1) VP16 activation domain.

[0218] Certain DNA-binding proteins contain separable domains thatfunction as transcriptional repressors. Approximately 20% of ZFPscontain a non-DNA-binding domain of about 90 amino acids that functionsas a transcriptional repressor (Thiesen, The New Biologist 2:363-374(1990); Margolin et al., PNAS 91:4509-4513 (1994); Pengue et al.,(1994), supra; Witzgall et al., (1994), supra). This domain, termed theKRAB domain, is modular and can be joined to other DNA-binding proteinsto block expression of genes containing the target DNA sequence(Margolin et al., (1994); Pengue et al., (1994); Witzgall et al.,(1994), supra). The KRAB domain has no effect by itself; it needs to betethered to a DNA sequence via a DNA-binding domain to function as arepressor. The KRAB domain has been shown to block transcriptioninitiation and can function at a distance of up to at least 3 kb fromthe transcription start site. The KRAB domain from the human KOX-1protein (Thiesen, The New Biologist 2:363-37 (1990)) was used for thestudies described here. This 64 amino acid domain can be fused to ZFPsand has been shown to confer repression in cell culture (Liu et al.,supra).

[0219] The VP16 protein of HSV-1 has been studied extensively, and ithas been shown that the C-terminal 78 amino acids can act as atrans-activation domain when fused to a DNA-binding domain (Hagmann etal., J. Virology 71:5952-5962 (1997)). VP16 has also been shown tofunction at a distance and in an orientation-independent manner. Forthese studies, amino acids 413 to 490 in the VP16 protein sequence wereused. DNA encoding this domain was PCR amplified from plasmid pMSVP16C+119 using primers with the following sequences:

[0220] (1) JVF24 CGCGGATCCGCCCCCCCGACCGATG (SEQ ID NO:21), and

[0221] (2) JVF25 CCGCAAGCTTACTTGTCATCGTCGTCCTTGTAGTCGCTGCCCCCACCGTACTCGTCAATTCC (SEQ ID NO:22)

[0222] The downstream primer, JVF25, was designed to include adownstream FLAG epitope-encoding sequence.

[0223] Three expression vectors were constructed for these studies. Thevectors are derived from pcDNA3.1 (+) (Invitrogen), and place the ZFPconstructs under the control of the cytomegalovirus (CMV) promoter. Thevector carries ampicillin and neomycin markers for selection in bacteriaand mammalian cell culture, respectively. A Kozak sequence for propertranslation initiation (Kozak, J. Biol. Chem. 266:19867-19870 (1991))was incorporated. To achieve nuclear localization of the products, thenuclear localization sequence (NLS) from the SV40 large T antigen(Pro-Lys-Lys-Lys-Arg-Lys-Val, SEQ ID NO:40) (Kalderon et al., Cell39:499-509 (1984)) was added. The insertion site for the ZFP-encodingsequence is followed by the functional domain sequence. The threeversions of this vector differ in the functional domain; “pcDNA-NKF”carries the KRAB repression domain sequence, “pcDNA-NVF” carries theVP16 activation domain, and “NF-control” carries no functional domain.Following the functional domain is the FLAG epitope sequence (Kodak) toallow specific detection of the ZFPs.

[0224] The vectors were constructed as follows. Plasmid pcDNA-HB wasconstructed by digesting plasmid pcDNA3.1(+) (Invitrogen, Carlsbad,Calif.) with HindIII and BamHI, filling in the sticky ends with Klenow,and religating. This eliminated the HindIII, KpnI, and BamHI sites inthe polylinker. The vector pcDNA3.1(+) is described in the Invitrogencatalog. Plasmid pcDNA-NKF was generated by inserting a fragment intothe EcoRI/Xhol sites of pcDNA-HB that contained the following: 1) asegment from EcoRI to KpnI containing the Kozak sequence including theinitiation codon and the SV40 NLS sequence, altogether comprising theDNA sequence GAATTCGCTAGCGCCACCATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCCATGGGGTAC (SEQ ID NO:23), where the EcoRI and KpnI sites are underlined;and 2) a segment from KpnI to XhoI containing a BamHI site, the KRAB-Abox from KOX1 (amino acid coordinates 11-53 in Thiesen, 1990, supra),the FLAG epitope (from Kodak/IBI catalog), and a HindIII site,altogether comprising the sequenceGGTACCCGGGGATCCCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGCAGCGACTACAAGGACGACGATGACAAGTAAGCTTCTCGAG (SEQ ID NO:24) where the KpnI, BamHI andXhoI sites are underlined.

[0225] The VEGF3a/1-KRAB effector plasmid was generated by inserting aKpnI-BamHI cassette containing the ZFP sequences into pcDNA-NKF digestedwith KpnI and BamHI. The VEGF1-KRAB and VEGF3a-KRAB effector plasmidswere constructed in a similar way except that the ZFP sequences werefirst cloned into the NLS-KRAB-FLAG sequences in the context of plasmidpLitmus 28 (New England Biolabs) and subsequently moved to theBamHI-XhoI sites of pcDNA3.1 (+) as a BglII-XhoI cassette, where theBglII site was placed immediately upstream of the EcoRI site (seeExample 4 for expression of these vectors).

[0226] The effector plasmids used in Example 5 were constructed asfollows. Plasmid pcDNA-NVF was constructed by PCR amplifying the VP16transactivation domain, as described above, and inserting the productinto the BamHI/HindIII sites of pcDNA-NKF, replacing the KRAB sequence.The sequence of the inserted fragment, from BamHI to HindIII, was:GGATCCGCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACG (SEQ ID NO:25)GCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGGCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGGGCAGCGACTACAAGGACGAC GATGACAAGTAAGCTT

[0227] VEGF1-VP16 and VEGF3a/1-VP16 vectors were constructed byinserting a KpnI-BamHI cassette containing the ZFP sequences intopcDNA-NVF digested with KpnI and BamHI.

[0228] The effector plasmids used in Example 6 were constructed asfollows. Plasmid NF-control was generated by inserting the sequenceGAATTCGCTAGCGCCACCATGGCCCCCAAGAAGAAGAGGAAGGTGGGAATCCATGGGGTACCCGGGGATGGATCCGGCAGCGACTACAAGGACGACGATGACA AGTAAGCTTCTCGAG (SEQID NO:26) into the EcoRI-XhoI sites of pcDNA-NKF, thereby replacing theNLS-KRAB-FLAG sequences with NLS-FLAG only.

[0229] VEGF1-NF and VEGF3a/1-NF were constructed by inserting aKpnI-BamHI cassette containing the ZFP sequences into NF-controldigested with KpnI and BamHI. CCR5-KRAB was constructed in the same wayas the VEGF KRAB vectors, except that the ZFP sequences were designed tobe specific for a DNA target site that is unrelated to the VEGF targets.

[0230] Finally, control versions of both the KRAB and VP16 expressionplasmids were constructed. Plasmid NKF-control was designed to expressNLS-KRAB-FLAG without zinc finger protein sequences; plasmid NVF-controlwas designed to express NLS-VP16-FLAG without ZFP sequences. Theseplasmids were made by digesting pcDNA-NKF and -NVF, respectively, withBamHI, filling in the ends with Klenow, and religating in order to placethe downstream domains into the proper reading frame. These plasmidsserve as rigorous controls for cell culture studies.

[0231] Mammalian cell expression and nuclear localization of the VEGFengineered ZFPs was demonstrated through immunofluorescence studies. 293(human embryonic kidney) cells were transfected with the expressionplasmid encoding the NLS-VEGF1-KRAB-FLAG chimera. Lipofectamine was usedas described below. After 24-48 hours, cells were fixed and exposed to aprimary antibody against the FLAG epitope. A secondary antibody labeledwith Texas Red was applied, and the cells were counter stained withDAPI. Texas Red staining was observed to consistently co-localize withthe DAPI staining, indicating that the ZFP being expressed from thisplasmid was nuclear localized.

Example 4

[0232] This Example demonstrates the use of transient co-transfectionstudies to measure the activity of the ZFP repressor proteins in cells.Such experiments involve co-transfection of ZFP-KRAB expression(“effector”) plasmids with reporter plasmids carrying the VEGF targetsites. Efficacy is assessed by the repression of reporter geneexpression in the presence of the effector plasmid relative to emptyvector controls.

[0233] The reporter plasmid system was based on the pGL3 fireflyluciferase vectors (Promega). Four copies of the VEGF target sites wereinserted upstream of the SV40 promoter, which is driving the fireflyluciferase gene, in the plasmid pGL3-Control to create pVFR1-4x. Thisplasmid contains the SV40 enhancer and expresses firefly luciferase tohigh levels in many cell types. Insertions were made by ligatingtogether tandem copies of the two complementary 42-bp oligonucleotides,JVF9 and JVF10, described in Example 2. Adaptor sequences were ligatedon, and the assembly was inserted into the MluI/BglII sites ofpGL3-Control. This resulted in the insertion of the following sequencebetween those sites:ACGCGTaagcttGCTAGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGAGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGAGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGAGCGAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGAGCGCTCAGaagcttAGATCT (SEQ ID NO:27)

[0234] The first six and last six nucleotides shown are the MluI andBglII sites; the lowercase letters indicate HindIII sites. The bindingsites for VEGF1 and VEGF3a are underlined.

[0235] The effector plasmid construction is described above. TheVEGF1-KRAB, VEGF3a-KRAB, and VEGF3a/1-KRAB expression vectors weredesigned to produce a fusion of the SV40 nuclear localization sequence,the VEGF ZFP, the KRAB repression domain, and a FLAG epitope marker allunder the control of the CMV promoter. The empty pcDNA3.1 expressionvector was used as a control (pcDNA).

[0236] All vectors were prepared using Qiagen DNA purification kits.Approximately 40,000 cells were seeded into each well of a 24-well plateand allowed to grow overnight in Dulbecco's Modified Eagle Medium(D-MEM) medium containing 10% fetal bovine serum at 37° C. with 5% CO₂.Cells were washed with PBS and overlayed with 200 μl of serum-freeD-MEM. Plasmids were introduced using lipofectamine (Gibco-BRL). Eachwell was transfected with about 0.3 μg of effector plasmid, 0.3 μg ofreporter plasmid, and 0.01 μg of plasmid pRL-SV40 (Promega) that hadbeen complexed with 6 μl of lipofectamine and 25 μl of D-MEM for 30 minat 37° C. Transfections were done in triplicate. After 3 hrs, 1 ml ofmedium containing 10% serum was added to each well. Cells were harvested40-48 hours after transfection. Luciferase assays were done using theDual Luciferase System (Promega). The third plasmid transfected,pRL-SV40, carries the Renilla luciferase gene and was co-transfected asa standard for transfection efficiency.

[0237] For the control reporter plasmid pGL3-Control (pGL3-C), thepresence or absence of the ZFP-KRAB expression plasmid does notinfluence the luciferase expression level. However, for pVFR1-4x, thereporter containing four copies of the VEGF target site, presence of theVEGF1 (9-bp-binding ZFP) or VEGF3a/1 (18-bp-binding ZFP) expressionplasmid reduces luciferase expression by a factor of 2-3 relative to theempty pcDNA vector control. The VEGF3a (9-bp-binding ZFP) expressionplasmid appears to exhibit little or no effect. These experimentsclearly demonstrate that a designed ZFP is capable of functioning in acell to repress transcription of a gene when its target site is present.Furthermore, it appears that a certain level of affinity is required forfunction; i.e., VEGF1 and VEGF3a/1, with K_(d)s of 10 μnM or less, arefunctional, whereas VEGF3a, with a K_(d) of 200 nM, is not.

[0238] A second reporter plasmid, pVFR2-4x, was constructed by removingthe four copies of the VEGF target sites using HindIII and insertingthem into the HindIII site of pGL3-Control (in the forward orientation).This places the target sites between the start site of transcription forthe SV40 promoter and the translational start codon of the luciferasegene. In co-transfection experiments similar to those described,approximately 3-4 fold repression of the luciferase signal was observedwith the VEGF1 -KRAB or VEGF3a/1-KRAB repressors relative to the pcDNAcontrols. This indicates that the repressors are active when boundeither upstream or downstream of the start of transcription.

Example 5

[0239] This Example demonstrates the use of transient co-transfectionstudies to measure the activity of the ZFP transcriptional activators incells. The experimental setup is similar to that of Example 4 exceptthat a different transfection method, a different cell line, and adifferent set of reporter and effector plasmids were used.

[0240] For activation experiments, a reporter was constructed labeledpVFR3-4x. This reporter contains the four copies of the VEGF targets,with the sequence shown above, at the MluI/BglII sites of plasmidpGL3-Promoter (Promega). This vector has been deleted for the SV40enhancer sequence and therefore has a lower basal level of fireflyluciferase expression. pVFR3-4x was constructed by swapping theKpnI/NcoI fragment of pVFR1-4x into the KpnI/NcoI sites ofpGL3-Promoter.

[0241] The effector plasmid construction is described above. TheVEGF1-VP16, VEGF3a-VP16, and VEGF3a /1-VP16 expression vectors weredesigned to produce a fusion of the SV40 nuclear localization sequence,the VEGF ZFP, the VP16 trans-activation domain, and a FLAG epitope tagall under the control of the CMV promoter. The empty pcDNA3 expressionvector was used as a control.

[0242] All vectors were prepared using Qiagen DNA purification kits.Approximately 40,000 cells were seeded into each well of a 24-well plateand allowed to grow overnight in D-MEM medium containing 10% fetalbovine serum at 37° C. with 5% CO₂. Cells were washed with serum-freeD-MEM and overlayed with 200 μl of the same. Plasmids were introducedusing a calcium phosphate transfection kit (Gibco-BRL) according to themanufacturer's instructions. Cells in each well were transfected with1.5 μg of reporter plasmid, 1.5 μg of effector plasmid, and 0.5 μg of anactin/β-gal plasmid. Plasmids were combined with 15 μl of CaCl₂ andbrought to 100 μl with dH₂O. 100 μl of HEPES solution was added dropwisewhile vortexing. The mix was incubated for 30 min at room temperature.The 200 μl of calcium phosphate-treated DNA was then added to the mediumin each well. Transfections were done in triplicate. After 5 hours, themedium was removed and 1 ml of medium containing 10% serum was added.Cells were harvested 40-48 hours after transfection. Luciferase assayswere done using the Dual-Light system (Tropix). The third plasmidtransfected, actinβ-gal, carries the β-galactosidase gene under thecontrol of the actin promoter and was co-transfected as a standard fortransfection efficiency. The β-galactosidase assays were also doneaccording to the manufacturer's protocol (Tropix).

[0243] For the control reporter plasmid, pGL3-Promoter (pGL3-P), thepresence or absence of the ZFP-VP16 expression plasmid does notsignificantly influence the luciferase expression level. For pVFR3-4x,the reporter containing four copies of the VEGF target site, presence ofVEGF1 (the 9-bp-binding ZFP) shows a very slight activation relative tothe empty pcDNA vector control. VEGF3a/1 (the 18-bp-binding ZFP)expression plasmid activates luciferase expression very substantially,showing about a 14-fold increase relative to pcDNA. These experimentsclearly demonstrate that a designed ZFP, when fused to the VP16activation domain, is capable of functioning in a cell to activatetranscription of a gene when its target site is present. Furthermore,these results clearly demonstrate that an 18-bp binding protein,VEGF3a/1, is a much better activator in this assay than a 9-bp bindingVEGF1 protein. This could be a result of the improved affinity ordecreased off-rate of the VEGF3a/1 protein.

[0244] A fourth VEGF reporter plasmid was constructed by cloning theKpnI/NcoI fragment of pVFR2-4x into pGL3-Promoter to create plasmidpVFR4-4x. Activation was observed in co-transfections using thisreporter in combination with effector plasmids expressing the VEGF1-VP16and VEGF3a/1-VP16 fusions. This indicates that these artificialtrans-activators are functional when bound either upstream or downstreamof the start of transcription.

[0245] These co-transfection data demonstrate that ZFPs can be used toregulate expression of reporter genes. Such experiments can serve as auseful tool for identifying ZFPs for further use as modulators ofexpression of endogenous molecular targets.

Example 6

[0246] This example demonstrates that a designed ZFP can repressexpression of an endogenous cellular gene that is in its normal genomicand chromatin context. Specifically, effector plasmids expressing VEGFZFPs fused to the KRAB repression domain were introduced into cells andwere shown to down-regulate the VEGF gene.

[0247] Eukaryotic expression vectors were constructed that fuse theVEGF3a/1 and the VEGF1 ZFPs to the SV40 NLS and KRAB, as described abovein Example 3. Transfections were done using Lipofectamine, acommercially available liposome preparation from GIBCO-BRL. All plasmidDNAs were prepared using Qiagen Midi DNA purification system. 10 μg ofthe effector plasmid was mixed with 100 μg of Lipofectamine (50 μl) in atotal volume of 1600 μl of Opti-MEM. A pCMVbeta-gal plasmid (Promega)was also included in the DNA mixture as an internal control fortransfection efficiency. Following a 30 minute incubation, 6.4 ml ofDMEM was added and the mixture was layered on 3×10⁶ 293 cells. Afterfive hours, the DNA-Lipofectamine mixture was removed, and fresh culturemedium containing 10% fetal bovine serum was layered on the cells.

[0248] Eighteen hours post transfection, the 293 cells were induced bytreatment with 100 μM DFX (desferrioxamine), resulting in a rapid andlasting transcriptional activation of the VEGF gene and also in agradual increase in VEGF mRNA stability (Ikeda et al., J. Biol. Chem.270:19761-19766 (1995)). Under routine culture conditions, 293 cellssecrete a low level of VEGF in the culture media. The cells were allowedto incubate an additional 24 hours before the supernatants werecollected for determination of VEGF levels by an ELISA assay.

[0249] In parallel experiments that demonstrated a similar level ofrepression, cell viability was monitored using the Promega Celltiter 96®Aqueous One Solution cell proliferation assay (Promega). After Dfxtreatment for 18 hours, 500 μl of the original 2 ml of media was removedand analyzed for VEGF expression, as described above. To evaluate cellviability, 300 μl of Promega Celltiter 96® Aqueous One Solution Reagentwas added to the remaining 1.5 ml. The cells were then incubated at 37°C. for approximately 2 hours. 100 μl from each well was transferred to a96-well plate and read on an ELISA plate reader at OD 490 nm. There wasno significant reduction in viability of cells expressing theVEGF3a/1-KRAB construct relative to those transfected with empty vectorcontrols, indicating that the VEGF repression observed was not due togeneralized cell death.

[0250] A 40-50-fold decrease in VEGF expression was noted in the DFXtreated cells transfected with VEGF3a/1-KRAB, an expression vectorencoding the 18 bp binding VEGF high affinity ZFP. A two-fold decreasein expression was observed when cells were transfected with VEGF1-KRAB,an expression vector encoding the 9 bp binding VEGF high affinity ZFP.No significant decrease in VEGF expression was observed in cells thatwere transfected with a non-VEGF ZFP (CCR5-KRAB) or NKF-control. Similarresults have been obtained in three independent transfectionexperiments.

[0251] In a separate experiment, the following results were obtained(data not shown). VEGF1-NF, which expresses the 9-bp-binding VEGF1 ZFPwithout a functional domain, showed no effect on VEGF gene expression. Asignificant reduction in VEGF expression was observed with VEGF3a/1-NF,which expresses the 18-bp binding protein without a functional domain.This result suggests that binding to the start site of transcription,even without a repression domain, interferes with transcription. Evenwhen fused to the KRAB domain, the VEGF3a ZFP is unable to affectexpression levels (plasmid VEGF3a-KRAB). However, VEGF1 fused to KRAB(VEGF1-KRAB) results in a dramatic decrease in expression. VEGF3a/1fused to KRAB (VEGF3a/1-KRAB) prevents expression of VEGF altogether.

[0252] These data indicate that a designed ZFP is capable of locatingand binding to its target site on the chromosome and preventingexpression of an endogenous cellular target gene. In particular, theresults indicate that ZFPs with a K_(d) of less than about 25 nM (e.g.,VEGF1 has an average apparent K_(d) of about 10 nM) provide dramaticdecreases in expression. In addition, the data demonstrate that the KRABfunctional domain enhances gene silencing. Because in this experimentthe introduction of the repressor occurs before the inducer of VEGF isadded (DFX), the data demonstrate the ability of a designed repressor toprevent activation of an already quiescent gene. In addition, theseresults demonstrate that a six-finger engineered ZFP (VEGF3a/1) withnanomolar affinity for its target is able to inhibit the hypoxicresponse of the VEGF gene when it binds a target that overlaps thetranscriptional start site.

Example 7

[0253] This Example demonstrates that a designed ZFP can activate theexpression of an endogenous cellular gene that is in its normal genomicand chromatin context. Specifically, effector plasmids expressing VEGFZFPs fused to the VP16 activation domain were introduced into cells andwere shown to up-regulate the VEGF gene.

[0254] Eukaryotic expression vectors were constructed that fuse theVEGF3a/1 and the VEGF1 ZFPs to the SV40 NLS and VP16, as described inExample 3. Transfections were done using Lipofectamine, a commerciallyavailable liposome preparation from GIBCO-BRL. All plasmid DNAs wereprepared using the Qiagen Midi DNA purification system. 10 μg of theeffector plasmid (containing the engineered ZFP) was mixed with 100 μgof Lipofectamine (50 μl) in a total volume of 1600 μl of Opti-MEM. ApCMVbeta-gal plasmid (Promega) was also included in the DNA mixture asan internal control for transfection efficiency. Following a 30 minuteincubation, 6.4 ml of DMEM was added and the mixture was layered on3×10⁶ 293 cells. After five hours, the DNA-Lipofectamine mixture wasremoved, and fresh culture medium containing 10% fetal bovine serum waslayered on the cells. One day later, fresh media was added and thesupernatant was collected 24 hours later for determination of VEGFlevels using a commercially available ELISA kit (R and D Systems).

[0255] For the three-fingered VEGF1-specific ZFP (VEGF1-VP16), a 7-10fold increase in VEGF expression was observed when compared to controlplasmid (NVF-control) and mock transfected cells. Similar results havebeen obtained in 5 independent experiments. It is important to note thatthe level of VEGF secretion in VEGF1-VP16 transfected cells wasequivalent or greater than the level in cells that have been treatedwith DFX. Introduction of VEGF3a/1-VP16 stimulated a more modestinduction of VEGF. This result is consistent with the finding in Example6, in which expression of the 18-bp binding protein without a functionaldomain prevented activation to a certain degree. This result suggestedthat the tight binding of this protein to the start site oftranscription interferes with activation.

[0256] These data indicate that a designed ZFP is capable of locatingand binding to its target site on the chromosome, presenting atranscriptional activation domain, and dramatically enhancing theexpression level of an endogenous gene. In particular, the resultsindicate that ZFPs with a K_(d) of less than about 25 nM (e.g., VEGF1has an average apparent K_(d) of about 10 nM) provide dramatic increasesin expression.

Example 8

[0257] To further substantiate the results in Examples 6 and 7, aribonuclease protection assay (RPA) was performed to correlate theincreased level of VEGF protein with an increase in VEGF mRNA levels(Example 7), and to correlate the decreased level of VEGF protein with adecrease in VEGF mRNA levels (Example 6).

[0258] RNA was isolated from the transfected cells using an RNAisolation kit (Pharmingen). Radiolabeled multi template probes, whichincluded a VEGF specific probe, were prepared by in vitro transcriptionand hybridized overnight at 56° C. to 5 μg of each of the RNAs from theexperimental and control transfected cells. The hybridization mixturewas treated with RNase and the protected probes were purified andsubjected to 5% denaturing polyacrylamide gel electrophoresis and theradioactivity was evaluated by autoradiography. 293 cells transfectedwith the VEGF1-VP16 had a 2-4 fold increase in the level of VEGF mRNAwhen compared to cells transfected with NVF-control (see Example 7 forexperimental details). The size of the protected probe was identical tothe size of the probe generated from the control human RNA provided as acontrol for RNA integrity.

[0259] In a separate experiment, the level of VEGF specific mRNA wasalso quantitated in cells that had been transfected with a VEGF-KRABeffector plasmid (see Example 6 for experimental details). The detailsof the transfection are described in Example 6. A dramatic decrease inthe level of VEGF mRNA was observed when cells were transfected with theVEGF3a/1-KRAB effector plasmid. No significant decrease in VEGF mRNA wasobserved when cells were transfected with NKF-control or a non-VEGFspecific ZFP (CCR5-5-KRAB and CCR5-3-KRAB, which recognize differentCCR5 target sites).

[0260] This experiment demonstrates that the increase in VEGF proteinobserved upon transfection with the VEGF1-VP16 chimeric transcriptionfactor is mediated by an increase in the level of VEGF mRNA. Similarly,the decrease in VEGF protein observed upon transfection with theVEGF3a/1-KRAB chimeric transcription factor is mediated by a decrease inthe level of VEGF mRNA.

Example 9 Identification of Inhibitors of the VEGFR2-mediated MitogenicSignal

[0261] Step 1: Production of cell lines

[0262] An ECV304-derived cell line that over-expresses VEGF-R2 and doesnot express VEGF-R1 is produced as follows. ZFP-repressors designedagainst VEGF-R1 are produced as described above. ZFP-activators designedto enhance VEGF-R2 also are generated. ECV304 cells are co-transfectedwith the two ZFP constructs; clones are picked for FACS analysis (R&DSystems, NFVE0) following drug-selection. A clone exhibiting a lack ofVEGF-R1 and an increase in VEGF-R2 on the membrane surface via FACSanalysis is expanded and the cell line is carried on for high throughput(HTP) screenings. This cell line is renamed VEGF-R1−/R2++.

[0263] A control cell line that constitutively represses VEGF-R1 andconditionally produces VEGF-R2 is produced as follows. ZFP-repressorsdesigned against VEGF-R2 are cloned into the T-Rex® (Invitrogen)plasmid. This and the VEGF-R1 ZFP-repressors are co-transfected intoECV304 cells. Once again, a stable clone is selected and analyzed for alack of VEGF-R1 and presence of VEGF-R2 via FACS analysis. The cells arethen treated with doxycycline and examined for a loss of VEGF-R2 viaFACS analysis. The cell line that does not express VEGF-R1 and shows aloss of VEGF-R2 following doxycycline treatment is carried forward forHTP screenings. This cell line is renamed VEGF-R1−/R2ind.

[0264] 2. Starting with VEGF-R1−/R2++, plate 10,000 cells/per well in a96-well plate. Because the final volume of the assay will be 100 μl,cells are plated in 50 μl of media.

[0265] 3. The test compound from the compound library is added (25 μl)to a final concentration of 30 nM and the plates are incubated for onehour. 25 μl of media is added without any compounds to one well to serveas the +VEGF control. To another well, add 0.075 ng in 25 μl of TGFβ(R&D Systems, 100-B-001) for a final concentration of 1 ng/ml, to serveas a VEGF-inhibitor control.

[0266] 4. Add VEGF (R&D Systems, 298-vs-005), 3 ng in 25 μl for a finalconcentration of 30 ng/ml, to each well. Incubate for 30 minutes.

[0267] 5. Assay for phosphorylated tyrosines within the cells bypermeabilizing the membrane. An alternative assay that measures proteintyrosine kinase activity is also available, skip to step 17.

[0268] 6. Aspirate off media. Fix cells with 1% formaldehyde for 30minutes.

[0269] 7. Wash cells with PBS.

[0270] 8. Add anti-phosphotyrosine antibody (Pierce, #29926), for afinal concentration of 5 μg/ml. Incubate for 1 hour.

[0271] 9. Wash cells with PBS containing 1% BSA and 0.05% NP-40.

[0272] 10. Add goat-anti-mouse-IgG-HRP conjugate (Pierce, #31430), 1:200dilution. Incubate for 1 hour.

[0273] 11. Wash cells as above.

[0274] 12. Perform HRP ELISA assay as described (Pierce, #32052).Identify compounds that reduce VEGF-stimulated phosphotyrosineproduction.

[0275] 13. Prepare for secondary screen by plating VEGF-R1−/R2ind-cellsas in step 2, this time preparing duplicate plates.

[0276] 14. To one plate, add doxycycline to a final concentration of 1μg/ml. Incubate overnight.

[0277] 15. To both plates, repeat steps 3-11.

[0278] 16. A positive hit shows reduced VEGF-stimulated phosphotyrosineproduction in the non-doxycycline treated cells. The ideal compoundbehaves the same in the doxycycline-treated cells, thus demonstratingthat it is not working on any systems other than the VEGF-R2-inducedpathway.

[0279] 17. Resuming from step 5. Aspirate off media. Solubilize cells byadding 200 μl of PTK extraction buffer (Promega, V6480) containing 1%Triton X-100) and incubate on ice for 10 minutes.

[0280] 18. Detach any residual cells and mix the cell suspension severaltimes by pipetting up and down several times. Transfer suspension into adeep well block and rock for 15 minutes at 4° C.

[0281] 19. Centrifuge the suspension at 100,000×g at 4° C. for 1 hourand save the supernatant.

[0282] 20. Continue with Step III.A.1 of the PTK Assay Protocol asdescribed by Promega (technical bulletin TB211).

Example 10 Identification of Stimulants of T Cell Proliferation That DoNot Cause Activation of the T Cells

[0283] Potassium channels play a critical role in modulating calciumsignaling of T lymphocytes. The two predominant potassium channelsexpressed in human T lymphocytes are Kv1.3, a channel that opens inresponse to changes in membrane potential, and KCa4, a channel activatedby elevations in intracellular calcium levels. Kv1.3 has been shown toplay a vital role in controlling T cell proliferation, whereas KCa4functions in T cell activation. Activation of the T cell then leads tothe release of IFN-γ and an immune response. The caveat to this pathwayis that KCa4 is up-regulated in response to known mitogenic andantigenic stimuli, thus, it has been difficult to identify compoundsthat can selectively allow for the proliferation of T lymphocytes but donot cause activation of the T cells. Although some KCa4 inhibitors havebeen identified, these inhibitors are not completely selective; they canalso inhibit other proteins such as the calcium-release-activated Ca2+(CRAC) channels, which also reside in T cells.

[0284] A ZFP-based inhibitor of the human hKCa4 gene was created usingthe methods of ZFP design described above. Its target sequence islocated at basepair 217 of the cDNA sequence (Genbank Accession #AE022797). The target sequence is 5′-GGGGAGGGC-3′ (SEQ ID NO:41). Theamino acid sequences (in single letter code) of the recognition helices(form −1 to +6) are as follows: F1=RSDHLAR (SEQ ID NO:42). F2=RSDNLAR(SEQ ID NO:43). F3=RSDHLSR (SEQ ID NO:44).

[0285] The resulting 3-finger ZFP, called ZFP3A, has a dissociationconstant of 0.3 nM for its target as measured by gel mobility shiftassay. A VP16 fusion to the ZFP3A DNA binding domain activates acotransfected reporter gene by greater than 5-fold.

[0286] A KRAB-ZFP3A chimeric construct to effect the inhibition of hKCa4gene expression was cotransfected into human erythroleukemic cells alongwith an expression vector encoding green fluorescent protein (GFP).Transfected cells were identified using a fluorescence microscope.Transfected cells were then examined for hKCa4 function by the patchclamp method. Ion channel function was determined by determining theslope conductance (nS). Untransfected controls had a value of 12.5(n=1). Cells transfected with an empty vector had a value of 10.0±2.5(n=4). Cells transfected with ZFP3A had a value of 1.0±0.1 indicatingalmost total loss of function.

[0287] The ZPF3A-transfected cells are further transfected with a secondZFP designed to activate Kv1.3 expression. The sequence encoding theactivator is cloned in the T-Rex™ (Invitrogen) plasmid. Stable clonesare selected using FACS analysis to identify those clones that have anincreased expression of Kv1.3. The clones are examined for changes inintracellular calcium fluctuations when treated with or withoutdoxycycline (1 μg/mL) using the FLIPR®I Flourometric Imaging PlateReader Systems (Molecular Dynamics). In the presence of doxycycline,there are minimal changes in the intracellular calcium fluctuations,whereas the non-doxycycline treated cells exhibit fluctuations similarto wild-type Jurkats. The cell line that has an increased expression ofKv1.3 and shows a loss of KCa4 following doxycycline treatment iscarried forward for HTP screenings. It is renamed Kv1.3⁺/KCa4^(+/−).

[0288] Kv1.3⁺/KCa4^(+/−) cells are plated, in duplicate, at 10,000 cells/well in 96-well dishes. Doxycycline is added to a final concentrationof 1 μg/mL to one plate, and both plates are incubated overnight.

[0289] The test compounds from the compound library are added to a finalconcentration of 30 nM to both plates and incubated for 24 hours. Foreach plate, a control well has 10 μg/mL of Concanavalin A (Con A,Amersham Pharmacia) to serve as the positive control for mitogenicactivity and T cell activation. A second control well has nothing addedto serve as the background control.

[0290] Four hours prior to harvesting, [³H]thymidine (1 μCi/well) isadded to each well.

[0291] The plates are spun to pellet the cells. Supernatants arecollected for use in IFN-γ ELISA (protocol and reagents from AmershamPharmacia).

[0292] Cells are resuspended in PBS and harvested onto glass fiberfilters. [³H]thymidine incorporation is measured in a scintillationcounter.

[0293] The cells not exposed to any mitogenic compound serve as thebaseline for [3H]thymidine uptake and IFN-γ secretion. The Con A controlexhibits an increased [³H]thymidine uptake in both doxycycline-treatedand non-treated cells; additionally, there is an increase in secretedIFN-γ in the non-treated cells but not in the doxycycline-treated cells.A positive hit is a compound that stimulates an increase in[³H]thymidine incorporation but not IFN-γ secretion in eitherdoxycycline-treated or non-treated cells.

Example 11 ZFP-Modulation of the Human Erythropoietin (EPO) Gene

[0294] In this example, the human erythropoietin (EPO) gene was used asan example of a molecular target whose expression can be modulated by aZFP. Accordingly, a stable cell line comprising an inducible ZFP wasestablished as described in Zhang et al. (2000) J. Biol Chem275:33850-33860. The cell line contained a stably-integrated fusion geneencoding a ZFP DNA binding domain designed to bind a target site 862nucleotides upstream of the human EPO gene (EPOZFP-862) fused to a VP16activation domain. Expression of this fusion gene was under the controlof tet transcriptional control sequences; and thus was inducible bydoxycycline.

[0295] The target site for the EPOZFP-862 protein was GCGGTGGCTC (SEQ IDNO: 36). The amino acid sequences of its recognition helices (betweenpositions −1 and +6 inclusive) were F1: QSSDLTR (SEQ ID NO: 37); F2:RSDALSR (SEQ ID NO: 38); and F3: RSDERKR (SEQ ID NO: 39). The EPOZFP-862zinc finger protein was designed in an SP-1 backbone, its gene wasassembled by PCR, purified as a fusion with the maltose-binding proteinand tested for its affinity for its target site as described supra.

[0296] Transient and stably transfected human embryonic kidney (HEK293)cells were generated as described in Zhang et al. (2000), supra.Briefly, the cells were grown in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal calf serum. To generate stable Tet-inducibleEPOZFP cell lines, the coding region from the pEPOZFP862 DNA wassubcloned into pcDNA4/TO (Invitrogen) using AflIII and HindIIIrestriction sites. The resulting pTO-EPOZFP862 construct was transfectedinto the T-Rex-293™ (Invitrogen) cell line using LipofectAMINE (LifeTechnologies, Inc.). After 2 weeks of selection in medium containingZeocin™ (Invitrogen), stable clones were isolated and analyzed fordoxycycline (Dox)-dependent activation of ZFP expression.

[0297] Transient transfection was carried out using LipofectAMINE. Celllysates were harvested 40 h after transfection, and luciferaseactivities were measured by the Dual-Light luciferase andβ-galactosidase reporter assay system (Tropix). To assay the activationof the endogenous chromosomal EPO gene, Northern analysis and Taqmananalyses of EPO mRNA were carried out as described in detail in Zhang etal. supra.

[0298] As shown in FIG. 2, the endogenous EPO gene was induciblyexpressed in response to synthesis of EPOZFP862. FIG. 2A shows EPOexpression and the Dox dose-response curve for stably transformed 293cells containing copies of the EPOZFP862 gene under control of aTet-responsive full-length CMV promoter. For this experiment,conditioned medium was harvested 48 h after the addition of Dox (at theconcentrations indicated in the figure) and analyzed by an EPO ELISAkit. FIG. 2B shows immunoblots of protein extracts from EPOZFP862 cellstreated with the indicated Dox concentrations, using an anti-Flagantibody. Extracts were prepared from cells 48 h after induction. FIG.2C shows RNA (Northern) blot analysis of EPO mRNA induced by EPOZFP862.EPO mRNA signals are shown for untransfected and EPOZFP862c-transfected293 cells (lanes 1 and 2, respectively) and for uninduced andDox-induced EPOZFP862 cells (lanes 3 and 4, respectively). The EPOprobed membrane was stripped and rehybridized with a ³²P-labeledriboprobe containing antisense fragments that hybridize to ZFP mRNA aswell as to the human β-actin gene that served as a loading control.

[0299] Thus, endogenous human EPO can be activated in response to eithertransient or stable expression of a ZFP targeted to the EPO gene.

[0300] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1 45 1 25 DNA Artificial Sequence Description of Artificial Sequencehuman VEGF 1 agcggggagg atcgcggagg cttgg 25 2 298 DNA ArtificialSequence Description of Artificial Sequence human SP-1 2 ggtacccatacctggcaaga agaagcagca catctgccac atccagggct gtggtaaagt 60 ttacggcacaacctcaaatc tgcgtcgtca cctgcgctgg cacaccggcg agaggccttt 120 catgtgtacctggtcctact gtggtaaacg cttcacccgt tcgtcaaacc tgcagcgtca 180 caagcgtacccacaccggtg agaagaaatt tgcttgcccg gagtgtccga agcgcttcat 240 gcgtagtgaccacctgtccc gtcacatcaa gacccaccag aataagaagg gtggatcc 298 3 99 PRTArtificial Sequence Description of Artificial Sequence ZFP-VEGF1 3 ValPro Ile Pro Gly Lys Lys Lys Gln His Ile Cys His Ile Gln Gly 1 5 10 15Cys Gly Lys Val Tyr Gly Thr Thr Ser Asn Leu Arg Arg His Leu Arg 20 25 30Trp His Thr Gly Glu Arg Pro Phe Met Cys Thr Trp Ser Tyr Cys Gly 35 40 45Lys Arg Phe Thr Arg Ser Ser Asn Leu Gln Arg His Lys Arg Thr His 50 55 60Thr Gly Glu Lys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met 65 70 7580 Arg Ser Asp His Leu Ser Arg His Ile Lys Thr His Gln Asn Lys Lys 85 9095 Gly Gly Ser 4 298 DNA Artificial Sequence Description of ArtificialSequence ZFP-VEGF3a 4 ggtacccata cctggcaaga agaagcagca catctgccacatccagggct gtggtaaagt 60 ttacggccag tcctccgacc tgcagcgtca cctgcgctggcacaccggcg agaggccttt 120 catgtgtacc tggtcctact gtggtaaacg cttcacccgttcgtcaaacc tacagaggca 180 caagcgtaca cacaccggtg agaagaaatt tgcttgcccggagtgtccga agcgcttcat 240 gcgaagtgac gagctgtcac gacatatcaa gacccaccagaacaagaagg gtggatcc 298 5 99 PRT Artificial Sequence Description ofArtificial Sequence ZFP-VEGF3a 5 Val Pro Ile Pro Gly Lys Lys Lys Gln HisIle Cys His Ile Gln Gly 1 5 10 15 Cys Gly Lys Val Tyr Gly Gln Ser SerAsp Leu Gln Arg His Leu Arg 20 25 30 Trp His Thr Gly Glu Arg Pro Phe MetCys Thr Trp Ser Tyr Cys Gly 35 40 45 Lys Arg Phe Thr Arg Ser Ser Asn LeuGln Arg His Lys Arg Thr His 50 55 60 Thr Gly Glu Lys Lys Phe Ala Cys ProGlu Cys Pro Lys Arg Phe Met 65 70 75 80 Arg Ser Asp Glu Leu Ser Arg HisIle Lys Thr His Gln Asn Lys Lys 85 90 95 Gly Gly Ser 6 29 DNA ArtificialSequence Description of Artificial Sequence VEGF-1 top 6 catgcatagcggggaggatc gccatcgat 29 7 29 DNA Artificial Sequence Description ofArtificial Sequence VEGF-1 bottom 7 atcgatggcg atcctccccg ctatgcatg 29 829 DNA Artificial Sequence Description of Artificial Sequence VEGF-3 top8 catgcatatc gcggaggctt ggcatcgat 29 9 29 DNA Artificial SequenceDescription of Artificial Sequence VEGF-3 bottom 9 atcgatgcca agcctccgcgatatgcatg 29 10 29 DNA Artificial Sequence Description of ArtificialSequence primer SPE7 10 gagcagaatt cggcaagaag aagcagcac 29 11 26 DNAArtificial Sequence Description of Artificial Sequence primer SPEamp1211 gtggtctaga cagctcgtca cttcgc 26 12 28 DNA Artificial SequenceDescription of Artificial Sequence primer SPEamp13 12 ggagccaaggctgtggtaaa gtttacgg 28 13 26 DNA Artificial Sequence Description ofArtificial Sequence primer SPEamp11 13 ggagaagctt ggatcctcat tatccc 2614 83 DNA Artificial Sequence Description of Artificial Sequence linkerXba-Sty 14 tctagacaca tcaaaaccca ccagaacaag aaagacggcg gtggcagcggcaaaaagaaa 60 cagcacatat gtcacatcca agg 83 15 39 DNA Artificial SequenceDescription of Artificial Sequence primer GB19 15 gccatgccgg tacccatacctggcaagaag aagcagcac 39 16 33 DNA Artificial Sequence Description ofArtificial Sequence primer GB10 16 cagatcggat ccacccttct tattctggtg ggt33 17 589 DNA Artificial Sequence Description of Artificial SequenceZFP-VEGF 3a/1 17 ggtacccata cctggcaaga agaagcagca catctgccac atccagggctgtggtaaagt 60 ttacggccag tcctccgacc tgcagcgtca cctgcgctgg cacaccggcgagaggccttt 120 catgtgtacc tggtcctact gtggtaaacg cttcacacgt tcgtcaaacctacagaggca 180 caagcgtaca cacacaggtg agaagaaatt tgcttgcccg gagtgtccgaagcgcttcat 240 18 196 PRT Artificial Sequence Description of ArtificialSequence ZFP-VEGF 3a/1 18 Val Pro Ile Pro Gly Lys Lys Lys Gln His IleCys His Ile Gln Gly 1 5 10 15 Cys Gly Lys Val Tyr Gly Gln Ser Ser AspLeu Gln Arg His Leu Arg 20 25 30 Trp His Thr Gly Glu Arg Pro Phe Met CysThr Trp Ser Tyr Cys Gly 35 40 45 Lys Arg Phe Thr Arg Ser Ser Asn Leu GlnArg His Lys Arg Thr His 50 55 60 Thr Gly Glu Lys Lys Phe Ala Cys Pro GluCys Pro Lys Arg Phe Met 65 70 75 80 Arg Ser Asp Glu Leu Ser Arg His IleLys Thr His Gln Asn Lys Lys 85 90 95 Asp Gly Gly Gly Ser Gly Lys Lys LysGln His Ile Cys His Ile Gln 100 105 110 Gly Cys Gly Lys Val Tyr Gly ThrThr Ser Asn Leu Arg Arg His Leu 115 120 125 Arg Trp His Thr Gly Glu ArgPro Phe Met Cys Thr Trp Ser Tyr Cys 130 135 140 Gly Lys Arg Phe Thr ArgSer Ser Asn Leu Gln Arg His Lys Arg Thr 145 150 155 160 His Thr Gly GluLys Lys Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe 165 170 175 Met Arg SerAsp His Leu Ser Arg His Ile Lys Thr His Gln Asn Lys 180 185 190 Lys GlyGly Ser 195 19 42 DNA Artificial Sequence Description of ArtificialSequence primer JVF9 19 agcgagcggg gaggatcgcg gaggcttggg gcagccgggt ag42 20 42 DNA Artificial Sequence Description of Artificial Sequenceprimer JVF10 20 cgctctaccc ggctgcccca agcctccgcg atcctccccg ct 42 21 25DNA Artificial Sequence Description of Artificial Sequence primer JVF2421 cgcggatccg cccccccgac cgatg 25 22 62 DNA Artificial SequenceDescription of Artificial Sequence primer JVF25 22 ccgcaagctt acttgtcatcgtcgtccttg tagtcgctgc ccccaccgta ctcgtcaatt 60 cc 62 23 61 DNAArtificial Sequence Description of Artificial Sequence SV40 NLS 23gaattcgcta gcgccaccat ggcccccaag aagaagagga aggtgggaat ccatggggta 60 c61 24 187 DNA Artificial Sequence Description of Artificial SequenceKRAB-FLAG 24 ggtacccggg gatcccggac actggtgacc ttcaaggatg tatttgtggacttcaccagg 60 gaggagtgga agctgctgga cactgctcag cagatcgtgt acagaaatgtgatgctggag 120 aactataaga acctggtttc cttgggcagc gactacaagg acgacgatgacaagtaagct 180 tctcgag 187 25 277 DNA Artificial Sequence Description ofArtificial Sequence VP16 FLAG 25 ggatccgccc ccccgaccga tgtcagcctgggggacgagc tccacttaga cggcgaggac 60 gtggcgatgg cgcatgccga cgcgctagacgatttcgatc tggacatgtt gggggacggg 120 gattccccgg ggccgggatt taccccccacgactccgccc cctacggcgc tctggatatg 180 gccgacttcg agtttgagca gatgtttaccgatgcccttg gaattgacga gtacggtggg 240 ggcagcgact acaaggacga cgatgacaagtaagctt 277 26 118 DNA Artificial Sequence Description of ArtificialSequence plasmid NF-control 26 gaattcgcta gcgccaccat ggcccccaagaagaagagga aggtgggaat ccatggggta 60 cccggggatg gatccggcag cgactacaaggacgacgatg acaagtaagc ttctcgag 118 27 204 DNA Artificial SequenceDescription of Artificial Sequence plasmid pGL3-control 27 acgcgtaagcttgctagcga gcggggagga tcgcggaggc ttggggcagc cgggtagagc 60 gagcggggaggatcgcggag gcttggggca gccgggtaga gcgagcgggg aggatcgcgg 120 aggcttggggcagccgggta gagcgagcgg ggaggatcgc ggaggcttgg ggcagccggg 180 tagagcgctcagaagcttag atct 204 28 5 PRT Artificial Sequence Description ofArtificial Sequence linker 28 Asp Gly Gly Gly Ser 1 5 29 5 PRTArtificial Sequence Description of Artificial Sequence linker 29 Thr GlyGlu Lys Pro 1 5 30 9 PRT Artificial Sequence Description of ArtificialSequence linker 30 Leu Arg Gln Lys Asp Gly Glu Arg Pro 1 5 31 4 PRTArtificial Sequence Description of Artificial Sequence linker 31 Gly GlyArg Arg 1 32 8 PRT Artificial Sequence Description of ArtificialSequence linker 32 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 33 9 PRTArtificial Sequence Description of Artificial Sequence linker 33 Leu ArgGln Arg Asp Gly Glu Arg Pro 1 5 34 12 PRT Artificial SequenceDescription of Artificial Sequence linker 34 Leu Arg Gln Lys Asp Gly GlyGly Ser Glu Arg Pro 1 5 10 35 16 PRT Artificial Sequence Description ofArtificial Sequence linker 35 Leu Arg Gln Lys Asp Gly Gly Gly Ser GlyGly Gly Ser Glu Arg Pro 1 5 10 15 36 10 DNA Artificial SequenceDescription of Artificial Sequence target sequence 36 gcggtggctc 10 37 7PRT Artificial Sequence Description of Artificial Sequence linker 37 GlnSer Ser Asp Leu Thr Arg 1 5 38 7 PRT Artificial Sequence Description ofArtificial Sequence linker 38 Arg Ser Asp Ala Leu Ser Arg 1 5 39 7 PRTArtificial Sequence Description of Artificial Sequence linker 39 Arg SerAsp Glu Arg Lys Arg 1 5 40 7 PRT Artificial Sequence Description ofArtificial Sequence SV40 large T antigen from nuclear localizationsequence 40 Pro Lys Lys Lys Arg Lys Val 1 5 41 9 DNA Artificial SequenceDescription of Artificial Sequence target sequence 41 ggggagggc 9 42 7PRT Artificial Sequence Description of Artificial Sequence recognitionhelix 42 Arg Ser Asp His Leu Ala Arg 1 5 43 7 PRT Artificial SequenceDescription of Artificial Sequence recognition helix 43 Arg Ser Asp AsnLeu Ala Arg 1 5 44 7 PRT Artificial Sequence Description of ArtificialSequence recognition helix 44 Arg Ser Asp His Leu Ser Arg 1 5 45 5 PRTArtificial Sequence Description of Artificial Sequence linker 45 Gly GlyGly Gly Ser 1 5

What is claimed is:
 1. A method of screening a compound for interactionwith a molecular target comprising (a) contacting a first cell with thecompound; (b) determining a first value of a property of the first cell,the property being responsive to the cell being contacted with thecompound; (c) contacting a second cell with the compound, wherein thesecond cell comprises an exogenous zinc finger protein that directly orindirectly modulates expression of the molecular target; (d) determininga second value of the property in the second cell, wherein a differencebetween the first and second values provides an indication ofinteraction between the compound and the molecular target.
 2. The methodaccording to claim 1, wherein the exogenous zinc finger protein directlymodulates expression of the molecular target.
 3. The method according toclaim 1, wherein the exogenous zinc finger protein is provided as apolypeptide.
 4. The method according to claim 1, wherein the exogenouszinc finger protein is provided as a polynucleotide encoding the zincfinger protein.
 5. The method of claim 1, wherein the exogenous zincfinger protein further comprises a functional domain.
 6. The method ofclaim 5, wherein the functional domain comprises an activation domain.7. The method of claim 6, wherein the activation domain is selected fromthe group consisting of VP16, p65 subunit of NF-kappa B, VP64 andligand-bound thyroid hormone receptor.
 8. The method of claim 5, whereinthe functional domain comprises a repression domain.
 9. The method ofclaim 8, wherein the repression domain is selected from the groupconsisting of KRAB, MBD-2B, v-ErbA, MBD3, unliganded TR and members ofthe DNMT family.
 10. The method according to claim 1, wherein themolecular target is involved in a signal transduction pathway.
 11. Themethod according to claim 10, wherein the compound is screened for itscapacity to block transduction of a signal through the molecular target.12. The method according to claim 1, wherein the second cell comprises asecond exogenous zinc finger protein.
 13. The method according to claim4, wherein the first and second cells are substantially identical exceptfor the polynucleotide encoding the zinc finger protein.
 14. The methodof claim 4, wherein the polynucleotide is stably transfected into thesecond cell.
 15. The method of claim 4, wherein the polynucleotideencoding the zinc finger protein is operably linked to an induciblepromoter.
 16. The method of claim 15, wherein expression of the zincfinger protein is induced in the second cell.
 17. The method of claim15, wherein the first and second cells comprise the polynucleotideencoding the inducible zinc finger protein.
 18. The method of claim 17,wherein expression of the zinc finger protein is not induced in thefirst cell.
 19. The method according to claim 1, wherein the moleculartarget is a protein.
 20. The method according to claim 1, wherein themolecular target is a cell surface receptor.
 21. The method according toclaim 1, wherein the expression level of the molecular target in thesecond cell is less than 25% of the expression level in the first cell.22. The method according to claim 1, wherein the expression level of themolecular target in the second cell is more than 125% of the expressionlevel in the first cell.
 23. The method according to claim 1, whereinthe expression level of the molecular target in the second cell is lessthan 5% of the expression level in the first cell.
 24. The methodaccording to claim 1, wherein the property is selected from the groupconsisting of growth, neovascularization, reporter expression andselectable marker expression.
 25. A method of screening a compound forinteraction with a molecular target, the method comprising: (a)isolating membranes from a cell comprising an exogenous zinc fingerprotein that modulates expression of the molecular target and (b)assaying interaction of the compound with the isolated membranes of step(a).
 26. The method of claim 25, wherein the molecular target isselected from the group consisting of a cell surface molecule and amembrane bound molecule.
 27. The method of claim 25, wherein theinteraction is assayed by measuring binding of the compound to theisolated membranes.
 28. The method of claim 27, wherein the compound isradiolabeled.
 29. A method of screening a compound for its effect on acellular process, the method comprising (a) providing a cell comprisingfirst and second polynucleotides, the first polynucleotide comprising afirst nucleic acid molecule encoding an exogenous zinc finger proteinoperably linked to a first transcriptional control element, wherein thefirst transcriptional control element is responsive to a moleculeinvolved in the cellular process, and the second polynucleotidecomprising a second nucleic acid molecule encoding a reporter orselectable marker, wherein expression of the reporter or selectablemarker is modulated by the exogenous zinc finger protein; (b) contactingthe cell of step (a) with the compound; and (c) assaying the expressionlevels of the reporter or selectable marker, wherein a change inexpression levels of the reporter or selectable marker indicates thatthe compound has an effect on the cellular process.
 30. The method ofclaim 29, wherein the cellular process comprises a signal transductionpathway.
 31. The method of claim 29, wherein the first polynucleotidefurther comprises sequences encoding a functional domain operably linkedto the zinc finger protein.
 32. The method of claim 31, wherein thefunctional domain is a repression domain.
 33. The method of claim 32,wherein the repression domain is selected from the group consisting ofKRAB, MBD-2B, v-ErbA, MBD3, unliganded TR, and members of the DNMTfamily.
 34. The method of claim 29, wherein the reporter or selectablemarker is selected from the group consisting of a direct reporter, anenzymatic reporter, a positive selection marker, a negative selectionmarker and combinations thereof.
 35. The method of claim 34, wherein thedirect reporter is a fluorescent protein.
 36. The method of claim 35,wherein the direct reporter is green fluorescent protein.
 37. The methodof claim 34, wherein the enzymatic reporter is selected from the groupconsisting of luciferase, beta-galactosidase, beta-glucuronidase,beta-lactamase, horseradish peroxidase, alkaline phosphatase andchloramphenicol acetyl transferase (CAT).
 38. The method of claim 34,wherein the positive selection marker is selected from the groupconsisting of neomycin resistance, G418 resistance, Zeocin® resistanceand hygromycin resistance.
 39. The method of claim 34, wherein thenegative selection marker is herpes simplex virus thymidine kinase(HSV-TK).
 40. A cell comprising an exogenous zinc finger protein thatdirectly or indirectly modulates expression of a molecular target. 41.The cell of claim 40, wherein the cell is comprises a polynucleotideencoding the zinc finger protein.
 42. The cell of claim 41, wherein thecell is stably transfected with the polynucleotide.
 43. The cell ofclaim 40, wherein the zinc finger protein directly modulates expressionof the molecular target.
 44. The cell of claim 43, wherein the zincfinger protein represses expression of the molecular target.
 45. Thecell of claim 43, wherein the zinc finger protein activates expressionof the molecular target.
 46. A cell comprising a polynucleotide encodinga ZFP and further comprising a polynucleotide encoding a reporter,wherein expression of the ZFP is regulated by a transcriptional controlelement that is responsive to a molecule involved in a cellular process;and wherein expression of the reporter is modulated by the ZFP.
 47. Thecell of claim 46, wherein the cellular process comprises a signaltransduction pathway.
 48. The cell of claim 46, wherein the firstpolynucleotide further comprises sequences encoding a functional domainoperably linked to the zinc finger protein.
 49. The cell of claim 48,wherein the functional domain is a repression domain.
 50. The cell ofclaim 49, wherein the repression domain is selected from the groupconsisting of KRAB, MBD-2B, v-ErbA, MBD3, unliganded TR, and members ofthe DNMT family.
 51. The cell of claim 46, wherein the reporter orselectable marker is selected from the group consisting of a directreporter, an enzymatic reporter, a positive selection marker, a negativeselection marker and combinations thereof.
 52. The cell of claim 51,wherein the direct reporter is a fluorescent protein.
 53. The cell ofclaim 52, wherein the direct reporter is green fluorescent protein. 54.The cell of claim 51, wherein the enzymatic reporter is selected fromthe group consisting of luciferase, beta-galactosidase,beta-glucuronidase, beta-lactamase, horseradish peroxidase, alkalinephosphatase and chloramphenicol acetyl transferase (CAT).
 55. The cellof claim 51, wherein the positive selection marker is selected from thegroup consisting of neomycin resistance, G418 resistance, Zeocin®resistance and hygromycin resistance.
 56. The cell of claim 51, whereinthe negative selection marker is herpes simplex virus thymidine kinase(HSV-TK).
 57. A kit for screening a compound for interaction with amolecular target, the kit comprising (a) a first cell according to claim40; (b) a second cell substantially identical to the first cell butlacking the exogenous zinc finger protein; (c) ancillary reagants; (d)instructions and (e) suitable containers.
 58. A kit for screening acompound for interaction with a molecular target, the kit comprising (a)a cell according to claim 46; (b) ancillary reagents; (d) instructionsand (e) suitable containers.